Treatment of Inflammation, Autoimmune, and Neurodegenerative Disorders with Immunosuppressive Tat Derivative Polypeptides

ABSTRACT

The present specification provides immunosuppressive Tat derivative polypeptides, compositions, and methods of using such polypeptides and compositions to treat an autoimmune disease, an inflammation-associated disease and/or a neurodegenerative disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/099,475, filed Dec. 6, 2013, which claims the benefit of U.S.provisional patent application 61/734,135 filed Dec. 6, 2012 and61/881,266 filed Sep. 23, 2013, the contents of each of which isincorporated by reference herein in their entirety.

FIELD

The present application is drawn to immunosuppressive HumanImmunodeficiency Virus (HIV) transactivator of transcription (Tat)derivative polypeptides for the treatment of diseases characterized byaberrant immune responses such as neurodegenerative and autoimmunediseases and inflammation-associated diseases.

BACKGROUND

The Human Immunodeficiency Virus (HIV) transactivator of transcription(Tat) mediates at least two independent activities, a receptor-mediatedtriggering event at the cellular surface and an intracellulartrans-activation activity that controls antigen-presenting cell (APC)differentiation. The receptor-mediated triggering event mediated by Tatis specific to APC, committing them for activation and differentiationinto highly immunosuppressive antigen presenting cell regulatorymacrophages (AReg) or into dendritic cells (DC) that stimulate specificcytotoxic T lymphocytes.

Antigen-presenting cells, macrophages and dendritic cells are criticalin the pathogenesis or response to a variety of diseases, disorders andundesired immune responses. Tat triggers monocytes to differentiate intoantigen-presenting macrophages expressing molecules that specificallysuppress the immune response to the presented antigen(s). In autoimmunediseases, certain of the body's own endogenous molecules are incorrectlyrecognized as foreign, resulting in extensive inflammation and tissuedamage. In one example, degradation of collagen type II into immunogenicpeptides can trigger rheumatoid arthritis (RA) in animals and has beenassociated with human RA. Considerable research has centered on reducingthe immune response to these proteins. The antigen-specificmacrophage-induced suppression attributed to Tat can be applied to thereduction of the undesired immune response to foreign and endogenousmolecules associated with inflammation and neurodegeneration.

Attempts to treat inflammation and autoimmune disorders have met withlimited success. This is due, in part, to the fact that the etiology ofinflammation and autoimmune disorders is a complex response based inpart on the various inflammation-inducing molecules and the multitude ofinflammation-mediating and -sensitizing molecules that appear to elicitinflammation via redundant mechanisms. Therefore, compounds,compositions, and methods that can treat inflammation, neurodegenerativedisease, or an autoimmune disorder would be highly desirable.

SUMMARY

The present specification discloses compounds, compositions, and methodsfor treating an individual suffering from diseases associated withaberrant immune responses, such as neurodegenerative or autoimmunedisorders or inflammation-associated diseases. This is accomplished byadministering a therapeutically effective amount of an immunosuppressiveTat derivative polypeptide or composition comprising such polypeptidesto an individual suffering from the disease. As disclosed herein, thedisclosed immunosuppressive Tat derivative polypeptides havedemonstrated immunosuppressive activity.

Disclosed herein are immunosuppressive trans-activator of transcription(Tat) derivative polypeptides comprising an amino acid sequencecomprising the following domains in the indicated order; a transcriptionfactor (TF) domain comprising a sequence from an immunosuppressive humanimmunodeficiency virus (HIV), SIV Tat protein, hairless or an artificialimmunosuppressive sequence; a cysteine-rich region from lentiviral Tator a defensin molecule; and a C-terminal region from a lentiviral Tatprotein.

In another embodiment, the immunosuppressive Tat derivative polypeptide,further comprising an arginine-rich domain from a lentiviral Tatprotein. In another embodiment, the TF domain further comprises a repeatsequence comprising (PVDPRLEPWKHPGSQP)_(n) at the N-terminus, whereinn=2-10. In another embodiment, at least one of the amino acids in the TFdomain is modified with a conservative amino acid substitution.

In another embodiment of the immunosuppressive Tat derivativepolypeptide, HIV is HIV-1 or HIV-2. In another embodiment, thelentiviral Tat is from HIV-1, HIV-2, SIV, FIV, BIV, or EIAV.

In another embodiment of the immunosuppressive Tat derivativepolypeptide, the TF domain comprises an amino acid sequence of one ofSEQ ID NOs: 36, 39, 44, 48, 50, 54, 59, 60, or 61. In anotherembodiment, the cysteine-rich domain comprises an amino acid sequence ofone of SEQ ID NOs:37, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 62,63, 64, or 70. In another embodiment, the C-terminal domain comprises anamino acid sequence of one of SEQ ID NOs:38, 42, 46, 47, 49, 52, 53, 68,or 71. In yet another embodiment, both the cysteine-rich region and theC-terminal region are from the same source and the amino acid sequenceof the combined cysteine-rich and C-terminal region is one of SEQ IDNOs:47, 49, or 53. In another embodiment, the immunosuppressive Tatderivative polypeptide has greater than 85% sequence identity, greaterthan 90% sequence identity, or greater than 95% sequence identity to oneof SEQ ID NOs:9-11, 13-35, or 69.

Also disclosed herein is a pharmaceutical composition comprising one ormore of an immunosuppressive Tat derivative polypeptides and apharmaceutically acceptable excipient. In another embodiment, theimmunosuppressive Tat derivative polypeptide has greater than 85%sequence identity to one or more of SEQ ID NOs:9-11, 13-35, or 69.

Further disclosed herein is a method of treating a disease characterizedby aberrant immune responses, the method comprising: administering atherapeutically effective amount of one or more of the immunosuppressiveTat derivative polypeptides to a subject in need thereof; and therebytreating the disease by suppressing the immune system.

Further disclosed herein is a method of increasing the expression of Fasligand (FasL) on antigen presenting cell regulatory macrophages (ARegs),the method comprising: administering a therapeutically effective amountof one or more of the immunosuppressive Tat derivative polypeptides to asubject; and thereby increasing the expression of FasL on the ARegs.

Further disclosed herein is a method of reducing inflammation, themethod comprising: administering a therapeutically effective amount ofone or more of the immunosuppressive Tat derivative polypeptides to asubject in need thereof; and thereby increasing reducing inflammation inthe subject.

In one embodiment of the methods disclosed herein, the immunosuppressiveTat derivative polypeptide has greater than 85% sequence identity to oneof SEQ ID NOs:9-11, 13-35, or 69.

In another embodiment of the methods, the treatment increases theexpression of Fas ligand on antigen presenting cell regulatorymacrophages (ARegs). In yet another embodiment, the ARegs are CD14+macrophages.

In another embodiment of the methods, the disease is an autoimmune,neurodegenerative or inflammation-associated disorder. In oneembodiment, the autoimmune disorder is an acute disseminatedencephalomyelitis (ADEM), an Addison's disease, an allergy, allergicrhinitis, an Alzheimer's disease, an anti-phospholipid antibody syndrome(APS), an arthritis, an asthma, an autoimmune deficiency syndrome, anautoimmune hemolytic anemia, an autoimmune hepatitis, an autoimmuneinner ear disease, a bullous pemphigoid, a celiac disease, a Chagasdisease, a chronic obstructive pulmonary disease (COPD), a diabetesmellitus type 1 (IDDM), an eczema, an endometriosis, a gastrointestinaldisorder, a Goodpasture's syndrome, a Graves' disease, a Guillain-Barrésyndrome (GBS), a Hashimoto's thyroiditis, a hidradenitis suppurativa,an idiopathic thrombocytopenic purpura, an inflammatory bowel disease,an inflammatory dermatologic disease, an interstitial cystitis, a lupus,a morphea, a multiple sclerosis (MS), a myasthenia gravis, a myopathy, anarcolepsy, a neuromyotonia, a pemphigus vulgaris, a pernicious anaemia,a primary biliary cirrhosis, a psoriasis, a recurrent disseminatedencephalomyelitis, a rheumatic fever, a schizophrenia, a scleroderma, aSjögren's syndrome, a skin disorder, a tenosynovitis, a uveitis, avasculitis, or a vitiligo. In another embodiment, the disease associatedwith inflammation is an acne, an acid reflux/heartburn, an allergy, anallergic rhinitis, an Alzheimer's disease, an appendicitis, anarteritis, an arthritis, an asthma, an atherosclerosis, an autoimmunedisorder, a balanitis, a blepharitis, a bronchiolitis, a bronchitis, abursitis, a cancer, a carditis, a celiac disease, a cellulitis, acervicitis, a cholangitis, a cholecystitis, a chorioamnionitis, achronic obstructive pulmonary disease (COPD), a cirrhosis, a colitis, aconjunctivitis, a cystitis, a common cold, a dacryoadenitis, a dementia,a dermatitis, a dermatomyositis, an eczema, an emphysema, anencephalitis, an endocarditis, an endometritis, an enteritis, anenterocolitis, an epicondylitis, an epididymitis, a fasciitis, afibrositis, a gastritis, a gastroenteritis, a gingivitis, aglomerulonephritis, a glossitis, a heart disease, a hepatitis, ahidradenitis suppurativa, a high blood pressure, an ileitis, an insulinresistance, an interstitial cystitis, an iritis, an ischemic heartdisease, a keratitis, a keratoconjunctivitis, a laryngitis, a lupus, amastitis, a mastoiditis, a meningitis, a metabolic syndrome (syndromeX), a migraine, a multiple sclerosis, a myelitis, a myocarditis, amyopathy, a myositis, a nephritis, a neuropathy, an obesity, anomphalitis, an oophoritis, an orchitis, an osteochondritis, anosteopenia, an osteoporosis, an osteitis, an otitis, a pancreatitis, aParkinson's disease, a parotitis, a pelvic inflammatory disease, apericarditis, a peritonitis, a pharyngitis, a phlebitis, a pleuritis, apneumonitis, a proctitis, a prostatitis, a psoriasis, a pulpitis, apyelonephritis, a pylephlebitis, a rheumatic fever, a rhinitis, asalpingitis, a sialadenitis, a sinusitis, a spastic colon, a stomatitis,a synovitis, a tendonitis, a tendinosis, a tenosynovitis, athrombophlebitis, a tonsillitis, a trigonitis, a tumor, an urethritis,an uveitis, a vaginitis, a vasculitis, or a vulvitis. In yet anotherembodiment, the neurodegenerative disease is Alexander disease, Alper'sdisease, Alzheimer's disease, amyloidoses, amyotrophic lateralsclerosis, anxiety, ataxia telangiectasia, attention deficit disorders,Canavan disease, central nervous system injuries, Charcot Marie Toothdisease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakobdisease, depression, encephalitis (e.g., bacterial, parasitic, fungal,or viral), Friedreich's ataxia frontotemporal dementia, hereditaryspastic paraparesis, Guillain-Barre syndrome (and its variants acutemotor axonal neuropathy, acute inflammatory demyelinatingpolyneuropathy, and Fisher syndrome), HIV/AIDS dementia complex,Huntington's disease, ischemic damage to the nervous system, Kennedy'sdisease, Krabbe disease, Lewy body dementia, Machado-Joseph disease,meningitis (e.g., bacterial, parasitic, fungal, or viral) multiplesclerosis, multiple system atrophy, neural trauma, e.g., percussivebrain damage, spinal cord injury and traumatic damage to the nervoussystem, a neuropathy such as e.g., chemotherapy-induced neuropathy,diabetes-associated neuropathy, and peripheral neuropathy, Parkinson'sdisease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateralsclerosis, prion disorders, progressive supranuclear palsy, Refsum'sdisease, Sandhoff disease, schizophrenia, Schilder's disease,spinocerebellar atrophies, Steele-Richardson-Olszewski disease, stroke,tabes dorsalis, or vascular dementia.

In yet another embodiment of the methods, the administration furthercauses the reduction of at least one symptom associated with theautoimmune disease, neurodegenerative disease, or disease associatedwith inflammation and therein the symptom is inflammation, fatigue,dizziness, malaise, elevated fever and high body temperature, extremesensitivity to cold in the hands and feet, weakness and stiffness inmuscles and joints, weight changes, digestive or gastrointestinalproblems, low or high blood pressure, irritability, anxiety, ordepression, infertility or reduced sex drive (low libido), blood sugarchanges, and depending on the type of autoimmune disease, an increase inthe size of an organ or tissue, or the destruction of an organ ortissue.

In another embodiment of the methods, the subject is notimmunocompromised. In another embodiment, the immune system of thesubject is not compromised as a result of the administration. In yetanother embodiment, as a result of the administration, secretion ofcytokines by ARegs is decreased.

In another embodiment of the methods, the immunosuppressive Tatderivative polypeptide is administered in a plurality of doses. Inanother embodiment, the immunosuppressive Tat derivative polypeptide isadministered daily, weekly, biweekly, monthly, or bimonthly. In yetanother embodiment, the administering step comprises a repetitiveadministration cycle wherein each cycle comprises administering aplurality of doses of the immunosuppressive Tat derivative polypeptidein a defined time period followed by a rest period and wherein the cycleis repeated a plurality of times.

Also disclosed herein is the use of one or more immunosuppressive Tatderivative polypeptides in the manufacture of a medicament for treatmentof a disease characterized by aberrant immune responses in a subject inneed thereof; wherein the one or more immunosuppressive Tat derivativepolypeptides are at least 85% identical to an immunosuppressive Tatderivative polypeptide of claim 1, and wherein administration of theimmunosuppressive Tat derivative polypeptide treats the disease bysuppressing the immune system in the subject.

Further disclosed herein is the use of one or more immunosuppressive Tatderivative polypeptides in the manufacture of a medicament forincreasing the expression of Fas ligand (FasL) on antigen presentingcell regulatory macrophages (ARegs) in a subject wherein the one or moreimmunosuppressive Tat derivative polypeptides are at lest 85% identicalto an immunosuppressive Tat derivative polypeptide of claim 1, whereinadministration of the immunosuppressive Tat derivative polypeptidesincreases the expression of FasL on the ARegs.

Further disclosed herein is the use of one or more immunosuppressive Tatderivative peptides in the manufacture of a medicament for reducinginflammation in a subject, wherein the one or more immunosuppressive Tatderivative polypeptides are at least 85% identical to animmunosuppressive Tat derivative polypeptide of claim 1, and whereinadministration of the immunosuppressive Tat derivative polypeptidesreduces inflammation in the subject.

In another embodiment of the use, the treatment increases the expressionof Fas ligand on antigen presenting cell regulatory macrophages (ARegs).In another embodiment, the ARegs are CD14+ macrophages. In anotherembodiment, secretion of cytokines by ARegs is decreased. In yet anotherembodiment, the subject is not immunocompromised. In yet anotherembodiment, the immune system of the subject is not compromised as aresult of the administration.

In yet another embodiment of the use, the immunosuppressive Tatderivative polypeptide has greater than 85% sequence identity to one ofSEQ ID NOs:9-11, 13-35, or 69.

In another embodiment of the use, the disease is an autoimmune,neurodegenerative or inflammation-associated disorder. In anotherembodiment, the autoimmune disorder is an acute disseminatedencephalomyelitis (ADEM), an Addison's disease, an allergy, allergicrhinitis, an Alzheimer's disease, an anti-phospholipid antibody syndrome(APS), an arthritis, an asthma, an autoimmune deficiency syndrome, anautoimmune hemolytic anemia, an autoimmune hepatitis, an autoimmuneinner ear disease, a bullous pemphigoid, a celiac disease, a Chagasdisease, a chronic obstructive pulmonary disease (COPD), a diabetesmellitus type 1 (IDDM), an eczema, an endometriosis, a gastrointestinaldisorder, a Goodpasture's syndrome, a Graves' disease, a Guillain-Barrésyndrome (GBS), a Hashimoto's thyroiditis, a hidradenitis suppurativa,an idiopathic thrombocytopenic purpura, an inflammatory bowel disease,an inflammatory dermatologic disease, an interstitial cystitis, a lupus,a morphea, a multiple sclerosis (MS), a myasthenia gravis, a myopathy, anarcolepsy, a neuromyotonia, a pemphigus vulgaris, a pernicious anaemia,a primary biliary cirrhosis, a psoriasis, a recurrent disseminatedencephalomyelitis, a rheumatic fever, a schizophrenia, a scleroderma, aSjögren's syndrome, a skin disorder, a tenosynovitis, a uveitis, avasculitis, or a vitiligo. In another embodiment, the disease associatedwith inflammation is an acne, an acid reflux/heartburn, an allergy, anallergic rhinitis, an Alzheimer's disease, an appendicitis, anarteritis, an arthritis, an asthma, an atherosclerosis, an autoimmunedisorder, a balanitis, a blepharitis, a bronchiolitis, a bronchitis, abursitis, a cancer, a carditis, a celiac disease, a cellulitis, acervicitis, a cholangitis, a cholecystitis, a chorioamnionitis, achronic obstructive pulmonary disease (COPD), a cirrhosis, a colitis, aconjunctivitis, a cystitis, a common cold, a dacryoadenitis, a dementia,a dermatitis, a dermatomyositis, an eczema, an emphysema, anencephalitis, an endocarditis, an endometritis, an enteritis, anenterocolitis, an epicondylitis, an epididymitis, a fasciitis, afibrositis, a gastritis, a gastroenteritis, a gingivitis, aglomerulonephritis, a glossitis, a heart disease, a hepatitis, ahidradenitis suppurativa, a high blood pressure, an ileitis, an insulinresistance, an interstitial cystitis, an iritis, an ischemic heartdisease, a keratitis, a keratoconjunctivitis, a laryngitis, a lupus, amastitis, a mastoiditis, a meningitis, a metabolic syndrome (syndromeX), a migraine, a multiple sclerosis, a myelitis, a myocarditis, amyopathy, a myositis, a nephritis, a neuropathy, an obesity, anomphalitis, an oophoritis, an orchitis, an osteochondritis, anosteopenia, an osteoporosis, an osteitis, an otitis, a pancreatitis, aParkinson's disease, a parotitis, a pelvic inflammatory disease, apericarditis, a peritonitis, a pharyngitis, a phlebitis, a pleuritis, apneumonitis, a proctitis, a prostatitis, a psoriasis, a pulpitis, apyelonephritis, a pylephlebitis, a rheumatic fever, a rhinitis, asalpingitis, a sialadenitis, a sinusitis, a spastic colon, a stomatitis,a synovitis, a tendonitis, a tendinosis, a tenosynovitis, athrombophlebitis, a tonsillitis, a trigonitis, a tumor, an urethritis,an uveitis, a vaginitis, a vasculitis, or a vulvitis. In yet anotherembodiment, the neurodegenerative disease is Alexander disease, Alper'sdisease, Alzheimer's disease, amyloidoses, amyotrophic lateralsclerosis, anxiety, ataxia telangiectasia, attention deficit disorders,Canavan disease, central nervous system injuries, Charcot Marie Toothdisease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakobdisease, depression, encephalitis (e.g., bacterial, parasitic, fungal,or viral), Friedreich's ataxia frontotemporal dementia, hereditaryspastic paraparesis, Guillain-Barre syndrome (and its variants acutemotor axonal neuropathy, acute inflammatory demyelinatingpolyneuropathy, and Fisher syndrome), HIV/AIDS dementia complex,Huntington's disease, ischemic damage to the nervous system, Kennedy'sdisease, Krabbe disease, Lewy body dementia, Machado-Joseph disease,meningitis (e.g., bacterial, parasitic, fungal, or viral), multiplesclerosis, multiple system atrophy, neural trauma, e.g., percussivebrain damage, spinal cord injury and traumatic damage to the nervoussystem, a neuropathy such as e.g., chemotherapy-induced neuropathy,diabetes-associated neuropathy, and peripheral neuropathy, Parkinson'sdisease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateralsclerosis, prion disorders, progressive supranuclear palsy, Refsum'sdisease, Sandhoff disease, schizophrenia, Schilder's disease,spinocerebellar atrophies, Steele-Richardson-Olszewski disease, stroke,tabes dorsalis, or vascular dementia.

In another embodiment of the use, the immunosuppressive Tat derivativepolypeptide further causes the reduction of at least one symptomassociated with the autoimmune disease, neurodegenerative disease, ordisease associated with inflammation and therein the symptom isinflammation, fatigue, dizziness, malaise, elevated fever and high bodytemperature, extreme sensitivity to cold in the hands and feet, weaknessand stiffness in muscles and joints, weight changes, digestive orgastrointestinal problems, low or high blood pressure, irritability,anxiety, or depression, infertility or reduced sex drive (low libido),blood sugar changes, and depending on the type of autoimmune disease, anincrease in the size of an organ or tissue, or the destruction of anorgan or tissue.

In another embodiment of the use, the immunosuppressive Tat derivativepolypeptide is administered in a plurality of doses. In anotherembodiment, the immunosuppressive Tat derivative polypeptide isadministered daily, weekly, biweekly, monthly, or bimonthly. In yetanother embodiment, the administering step comprises a repetitiveadministration cycle wherein each cycle comprises administering aplurality of doses of the immunosuppressive Tat derivative polypeptidein a defined time period followed by a rest period and wherein the cycleis repeated a plurality of times.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-B depicts fluorescence activated cell sorter (FACS) analysis ofthe results of Tat activation of monocytes. Human peripheral bloodmonocytes were committed to differentiate into dendritic cells (DC)through five days of culture in GM-CSF and IL-4. Committed DCs werecultured overnight either in medium alone (control), lipopolysaccharide(LPS), or Tat, after which they were stained with an anti-CD86 antibodyand analyzed by FACScan for CD86, a specific marker of DC activation,induction (FIG. 1A) or generalized activation (FIG. 1B, enlargement intobox R2, shown for Tat-stimulated cells).

FIG. 2 depicts the enhancement of antigen-specific activation ofcytotoxic T lymphocytes (CTL) by Tat*-antigen (Ag) complexes. CTLactivity was quantitated as the number of γ-interferon-secretingspot-forming colonies (SFC)/10⁶ plated cells using ELISPOT assays.

FIG. 3 depicts median fluorescence of monocytes, cultured for six dayseither with no stimulus (0), tumor necrosis factor-alpha (TNF-α), LPS,decreasing concentrations of C-Tat (conventional nativeimmunosuppressive Tat from HIV), or oxidized C-Tat (ox-C-Tat) andstained with an anti-Fas ligand (FasL) monoclonal antibody (Mab)followed by a fluoresceinated goat anti-mouse polyclonal antibody.

FIG. 4A-B depict antibody titer to an immunogenic antigen in thepresence of the immunosuppressive Tat (PT) or with non-immunosuppressiveox-Tat* (Ag) after two weeks (FIG. 4A) or six weeks (FIG. 4B).

FIG. 5 depicts FACS analysis of mouse peritoneal macrophages that wereisolated either after in vivo thioglycolate stimulation(stimulated+adjuvant) or without in vivo stimulation (resting). Mouseperitoneal macrophages were cultured for five days either in the absenceof additional stimulation (C), with LPS or with Tat. Activation wasdetermined as percent enlarged cells (M1 fraction).

FIG. 6 depicts stable suppression of antigen-stimulated T lymphocytes byTat-Ag complexes two weeks after immunization with immunosuppressiveTat.

FIG. 7 depicts the antigen-specificity of Tat suppression. Mice wereimmunized at day 0 and boosted at day 7 with an adjuvant emulsioncontaining either Tat (Ag+Tat), or with Ag alone as control. At day 14,draining lymph node cells were harvested and stimulated with eitherspecific or non-specific antigen and proliferation measured by ³Hthymidine uptake (CPM) after four days of culture.

FIG. 8 depicts FACS analysis of human peripheral blood monocytescultured for four days in control medium (control), or medium containingTat or LPS. Harvested cells were doubly stained with a fluoresceinatedanti-FasL Mab (αFasL-FITC) and with an anti-CD14 rhodamine labeled Mab.Cells were analyzed by FACScan for activation (forward scatter), CD14expression (% macrophages, R2), and for induction of FasL (MFI). The Tcell population is labeled R1.

FIG. 9A-B depicts the regulatory and immunosuppressive characteristicsof Tat-activated macrophages. (FIG. 9A) Human peripheral bloodmononuclear cells (PBMC) from one individual (PBMCs #3) cultured forfive days in either medium with tetanus antigen (Ag), antigen with thefurther addition of Tat (Ag+Tat) or Ag with Tat and recombinant sFasprotein (Ag+Tat+sFas). The results are graphed as stimulation index(mean cpm stimulated culture/mean cpm medium control). (FIG. 9B)Proliferation of PBMCs cultured 6 days with either tetanus or Candidaantigen alone (Ag), compared with cultures in which Tat (Ag+Tat), or Tatand the antagonistic anti-Fas antibody, ZB4, were added (Ag+Tat+αFas).

FIG. 10 depicts stimulation of human monocytes with Tat derivatives.

FIG. 11 depicts a dose-response curve of stimulation of human monocyteswith Tat derivatives.

FIG. 12A-C depicts staining of Jurkat cells with Hoechst-NucBlue andAlexa488-labeled Tat derivative SEQ ID NO:9 (FIG. 12A), Alexa488-labeledTat derivative SEQ ID NO:11 (FIG. 12B), or control (no Tat derivative,FIG. 12C). Left column represents nuclear staining with Hoechst-NucBlue,center column represents Alexa488 staining, and the right column depictsa merge of the left and center images.

FIG. 13A-C depicts staining of U937 cells with Hoechst-NucBlue andAlexa488-labeled Tat derivative SEQ ID NO:9 (FIG. 13A), Alexa488-labeledTat derivative SEQ ID NO:11 (FIG. 13B), or control (no Tat derivative,FIG. 13C). Left column represents nuclear staining with Hoechst-NucBlue,center column represents Alexa488 staining, and the right column depictsa merge of the left and center images.

FIG. 14A-C depicts activation of human peripheral blood mononuclearcells (PBMC), as measured by FasL expression, with no stimulus (FIG.14A), LPS (FIG. 14B), or Tat derivative SEQ ID NO:9 (FIG. 14C).

FIG. 15 depicts proliferation of human CD14+ PBMCs, as measured by cellsurface CD14 expression, with no stimulus, LPS, or Tat derivative SEQ IDNO:9.

FIG. 16 depicts the percent CD14+ cells in three day cultures of PBMCstreated with LPS, Tat derivative SEQ ID NO:9, or with no stimulus.

FIG. 17A-E depicts PBMCs expressing both FasL and CD14 after treatmentwith TNF-alpha (FIG. 17B), LPS (FIG. 17C), Tat derivative SEQ ID NO:9(FIG. 17D), or with no stimulation (FIG. 17A) after five days inculture. FIG. 17E depicts the data from FIGS. 27A-D in graphical format.

DESCRIPTION

The present specification relates to compounds, compositions, andmethods for treating an individual suffering from diseases associatedwith aberrant immune responses, such as autoimmune disorders includingneurodegenerative disorders, or inflammation-associated disorders. Thisis accomplished by administering a therapeutically effective amount ofan immunosuppressive Tat derivative polypeptide or compositioncomprising such polypeptides to an individual suffering from thediseases. As disclosed herein, the disclosed immunosuppressive Tatderivative polypeptides have anti-inflammatory activities. Theimmunosuppressive derivative peptides within the scope of the presentdisclosure are immunosuppressive.

As used herein the term “aberrant immune responses” refers to increased,undesirable, excessive, or inappropriate immune responses in which theimmune response to antigens, such as self antigens, is increased suchthat inflammation and/or autoimmune disease or neurodegenerative diseaseis seen. Aberrant immune responses, as used herein, are characterized byan immune cascade resulting in destruction of the body's tissue.Typically, an aberrant immune result is not seen in a normal response toinfection but can be triggered by infection.

The HIV Tat protein is a variable RNA binding peptide of 86-110 aminoacids in length that is encoded on two separate exons of the HIV genome.Based on molecular analysis, the Tat protein (SEQ ID NO:2) encodesdistinct and linked peptide activities. This present disclosuredescribes polypeptide compositions that are derivatized from thecanonical HIV-1 Tat structure in at least at the first or amino terminalportion, in a manner to enhance the immunotherapeutic potential of thepolypeptide. The amino terminal portion of Tat includes a short peptideregion from a nuclear transcription factor (TF) typically flanked byproline residues. This region determines, at least in part, howstimulatory or how suppressive the Tat polypeptide is for cells of theimmune system, particularly innate immune cells such as dendritic cells(DC) and macrophages (antigen-presenting cells or APCs). Consequently,it is predicted that modifications to the TF region can render thepolypeptides more active in the therapy of disease.

Previous studies determined that HIV Tat is immunosuppressive in themajority of human HIV strains. However, in long-term non-progressors(LTNP), a subset of HIV-infected individuals with high viral loads whodo not have a significant reduction in T4 cells and do not progress toAcquired Immunodeficiency Syndrome (AIDS), the HIV Tat protein isimmunostimulatory. The Tat protein found in LTNP is capable oftrans-activating viral RNA; however, LTNP Tat (designated herein afteras “IS-Tat” for immunostimulatory Tat) does not induce apoptosis in T4cells or macrophages and is not immunosuppressive. Moreover, T4 cellsinfected ex vivo with HIV isolated from LTNP (such cell lines aredesignated “Tat TcL”) overexpress IS-Tat proteins, often to the virtualexclusion of other viral proteins, that are strongly growth promotingrather than pro-apoptotic. The tat genes cloned from these Tat TcLsreveal sequence variations in two tat regions, at the amino terminus andwithin the first part of the second exon.

Based on molecular analysis, the HIV Tat protein (SEQ ID NO:2) containsthree distinct regions of interest. The first region of interest is thetransduction domain at the amino terminus of Tat (amino acids 3-19). Asecond region of interest is a cysteine-rich ligand binding domain(amino acids 22-37) which contains seven conserved cysteines. A thirdregion of interest is the membrane translocation sequence (MTS) whichencompasses amino acids 47-57.

The proline rich stretch near the amino terminus (amino acids 3-19) ofHIV-1 and HIV-2 Tat within the transduction domain is an SH3 bindingdomain having significant homology to the SH3-binding domain of themouse hairless (hr) gene. The SH3 binding domain presents an importanttarget for pharmaceutical development with the potential for thediagnosis, prophylaxis and treatment of undesirable cellular processessuch as autoimmunity.

Unexpectedly, mice expressing the hr gene mutation develop an AIDS-likesyndrome characterized by poor CTL function, a shift in helper Tlymphocytes from those regulating cell-mediated immunity (TH1) to thoseregulating antibody-mediated immunity (TH2), and increasedsusceptibility to chemical and ultraviolet light-induced skin cancers.Additionally, variants of Tat are found in retrovirus-infected monkeyswhich do not develop immunodeficiency and that do not have epidemicinfection. However, these variant Tat do not have the SH3 binding domainand instead substitute a different sequence, also set off by prolines ateither end of the sequence, into the transduction domain. Therefore, theSH3 binding domain is central to the immunosuppressive activity of Tat.Genetic data indicates the SH3 binding domain regulates monocytedifferentiation into antigen-presenting cell regulatory macrophages(ARegs). In Tat proteins which do not contain this SH3 domain, or thisdomain is mutated, monocyte differentiation is directed into DCs whichstimulate CTL responses

The MTS region permits Tat to freely traffic across the endosomalmembrane into the cytoplasm following receptor binding, where ittransactivates gene expression, including but not restricted to, genesof HIV-1. The MTS has been wrongly assumed to facilitate Tat entranceinto the cell, which it can only accomplish at high concentrations thathave been impossible to attain in vivo.

Unlike the current immunosuppressive therapies, the disclosed Tat-basedcompositions, the immunosuppressive Tat derivative polypeptides, havethe potential to suppress antigen-specific immune responses withoutimmunocompromising the patient. They preserve the immunosuppressiveactivity of conventional HIV Tat in the absence of the virus andtherefore the immunosuppressive Tat derivatives modulate the specificityof the immune response, a key component to the bodies natural defenseThis is particularly important when chronic immunosuppressive therapy isneeded, such as in autoimmune, neurodegenerative, orinflammation-associated diseases.

The immunosuppressive effects of Tat are mediated by macrophages. Whenstimulated by Tat, either by natural HIV-1 infection or by Tat uptake,macrophages induce the Fas ligand (FasL), which in turn induces theprogrammed cell death (apoptosis) of antigen-reacting, Fas-expressinghelper T cells (FIG. 3). Tat enhances the viability of cultured murinemacrophages as long as the macrophages were first activated in vivocompared with no prior activation and stimulated with relatively highconcentrations of Tat. By comparison, LPS promotes the viability ofmurine macrophages independently from in vivo stimulation, and at thesame concentration effective for human macrophages. Certain of theTat-based compositions disclosed herein produce a stable suppression ofmouse lymphocyte proliferation and may also serve to suppress anantigen-specific immune response to a variety of antigens.

The macrophages responsible for these responses have been identified asantigen presenting cell regulatory macrophages (ARegs). ARegs are alsoknown as “alternatively activated” macrophages. ARegs are stablemacrophages expressing FasL and secreting the cytokines IL-10 and IL-6.AReg are stable and respond in an autocrine and paracrine manner tothese two cytokines, as well as in a paracrine manner to IL-4. Thesecytokines accumulate and switch the immune response from TH1 (based onhelper T lymphocytes) to TH2 (based on suppressive T lymphocytes). Asthese cytokines build up, they overwhelm and suppress the immuneresponse and explain why immune responses are normally self-limiting inan antigen-specific manner.

An unexpected observation is that 1,000 fold lower concentrations of Tat(500 pM) trigger this effect on the macrophages, as compared with theconcentration required to initiate direct apoptosis of CD4+ T cells(approximately 500 nM). Therefore, at concentrations of Tat achievableas a systemically administered immunomodulator, the macrophage effectwill preferentially occur over the T cell effect.

The Tat-mediated antigen-specific suppression is mediated throughtrans-(intracellular) activation of a CD14+ FasL+ macrophage.Tat-activated macrophages are immunosuppressive ARegs. At lowconcentrations of Tat (50 nM), Tat-induced immunosuppression was notonly fully reversed by the addition of soluble Fas, but under theseconditions, Tat actually became slightly stimulatory (relative toantigen treatment alone). Antibodies to FasL reversed Tatimmunosuppression of tetanus responses and enhanced the Candida responserelative to Tat treatment alone. Suppression could be fully reversed(>95% of control) with the further addition of anti-IL-10 and anti-IL-6antibodies to the cultures, both cytokines deriving from macrophagesunder these culture conditions. A portion of Tat-inducedimmunosuppression is contributed by induction of FasL, although otherTat-induced factors can participate in suppressing T cell proliferativeresponses, especially at higher concentrations of Tat.

The complete amino acid sequence of HIV-1 Tat encoded by exons 1 and 2of the Tat gene is listed below:

ATG GAG CCC GTG GAC CCT CGC CTG GAG CCC TGG AAG CAC CCG GGC AGCMet Glu Pro Val Asp Pro Arg Leu Glu Pro Trp Lys His Pro Gly Ser1               5                   10/30               15CAG CCC AAG ACC GCC TGC ACC ACA TGT TACT GC AAG AAG TGC TGC TTCGln Pro Lys Thr Ala Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe            20/60               25                  30/90CAC TGC CAG GTG TGC TTC ACC AAG AAG GCC TTG GGC ATC AGC TAC GGCHis Cys Gln Val Cys Phe Thr Lys Lys Ala Leu Gly Ile Ser Tyr Gly        35                  40/120              45CGC AAG AAG CGC CGG CAG CGC CGC CGG GCC CCT GAG GAC AGC CAG ACCArg Lys Lys Arg Arg Gln Arg Arg Arg Ala Pro Glu Asp Ser Gln Thr    50/150              55                  60/180CAC CAG GTG AGC CCT CCC AAG CAG CCC GCT CCA CAG TTC CGC GGC GACHis Gln Val Ser Pro Pro Lys Gln Pro Ala Pro Gln Phe Arg Gly Asp65                  70/210              75                  80/240CCT ACC GGT CCC AAG GAG AGC AAG AAG AAG GTG GAG CGC GAG ACC GAGPro Thr Gly Pro Lys Glu Ser Lys Lys Lys Val Glu Arg Glu Thr Glu                85                  90/270              95ACC CAT CCC GTC GAC SEQ ID NO: 1 Thr His Pro Val Asp SEQ ID NO: 2            100/300

Tat has a proline (P) rich segment near the amino terminus (amino acids3-19, underlined above). This highly conserved region of HIV-1 Tat is acanonical SH3 binding domain also referred to herein as a nucleartranscription factor (TF) domain. The mouse hairless (hr) gene also hasan SH3 binding motif (TF, amino acids 176-196 of hr [SEQ ID NO:72]).Homology exists between the human Tat SH3 binding domain (SEQ ID NO:4)and the SH3 binding domain of the mouse hr gene:

Human 3 Pro Val Arg Pro Asn Leu Glu Pro Trp Lys His Pro Gly Ser Gln Pro18 Mouse 180Pro Leu Thr Pro Asn ------- Pro Trp Val Tyr Ser Gly Ser Gln Pro 193

Variants of Tat found in simian retroviruses, which do not causeimmunodeficiency, do not have an SH3 binding domain but instead have thefollowing proline-flanked sequence:

(SEQ ID NO: 3) Pro Leu Arg Glu Gln Glu Asn Ser Leu Glu Ser SerAsn Glu Arg Ser Ser Cys Ile Leu Glu Ala Asp Ala Thr Thr Pro

The human equivalent of the simian sequence (SEQ ID NO:3) above is:

(SEQ ID NO. 4) Ser Asn Glu Arg Ser Ser Cys Glu Leu Glu Val

Another region of interest is a cysteine-rich proposed ligand bindingdomain (amino acids 22-37) which contains seven cysteines.

(SEQ ID NO: 5) Cys Thr Thr Cys Tyr Cys Lys Lys Cys Cys Phe HisCys Gln Val Cys

Derivatives of Tat, generated through modulating the signal transductionmotif defined by the SH3 binding domain, drive differentiationpredominantly to dendritic cells or immunosuppressive AReg. AReg arealso critical contributors to invasion of gastric, pancreas, and ductalinfiltrating breast tumors, as well as components of tolerance in organtransplantation. The two external prolines at positions 3 and 18flanking the SH3 domain are maintained in order to facilitate the properstructure for SH3 binding. In addition, the transduction domain from anon-immunosuppressive human variant Tat, or the domain from the hrmutation, can replace amino acids 3-19 of Tat, although the hr sequenceis predicted to increase suppression. In addition, the stimulatorysimian form of Tat (SEQ ID NO:3), or its human equivalent sequence (SEQID NO:4), can be substituted at this domain. Additional chemicalmodifications, such as ox-Tat (chemically oxidized Tat as disclosed inUS 2006/0160183, incorporated by reference for all it contains regardingTat derivatives), can be used for stimulation of dendritic/CTLresponses.

In one embodiment disclosed herein, the immunosuppressive Tat derivativecomprises a Tat peptide in which amino acids 3-19 are altered. Thesealterations include replacement of individual amino acids with alternateamino acids or replacement of all of amino acids 3-19 with anothersequence. Tat peptides suitable for use in constructing the disclosedimmunosuppressive Tat derivatives include Tat from HIV-1 variants, HIV-2variants, and SIV variants. SIV variants can be from any species ofprimate that is infected with SIV as listed in Table 4. Also useful areimmunosuppressive lentiviral Tat sequences from non-primate speciesincluding, but not limited to, feline (Tat from feline immunodeficiencyvirus, FIV), bovine, (Tat from bovine immunodeficiency virus, BIV), orequine (Tat from equine infectious anemia virus, EIAV). For the purposesof the instant disclosure, the term “variants” refers to peptidescorresponding to the sequence of different strains, naturally occurringor mutated, of the indicated viruses.

SH3 binding proteins contain a series of internal prolines required fornuclear transcription factor (TF) function. In certain embodiments, theinternal prolines are each substituted by alanine, rendering theSH3-binding site inactive.

Exemplary immunosuppressive Tat derivatives within the scope of thepresent disclosure are presented in Table 1. In general, animmunosuppressive Tat derivative polypeptide for the treatment ofinflammation, autoimmune disorders, and neurodegeneration comprisesthree regions. The first region is a derivatized TF, the second regionis a cysteine-rich region, and the third region is a C-terminal Tatregion.

The TF region, cysteine-rich region, and a C-terminal region arearranged in the Tat derivative polypeptide in that order. The TF regionmay be derived from a source including, but not limited to, HIV-1 Tat,HIV-2 Tat, SIV Tat, the hairless gene, or an artificialimmunosuppressive sequences. The cysteine-rich region may be from alentivirus Tat, or cysteine rich defensin molecule. The C-terminalregion may be derived from lentivirus Tat. Additionally, the Tatderivative C-terminal regions may contain therein an arginine-richregion from HIV-1 Tat, HIV-2 Tat, or SIV Tat (also referred to as themembrane translocation sequence).

As used herein, the term “defensin molecule” refers to smallcysteine-rich cationic proteins found in both vertebrates andinvertebrates. Defensins comprise 18-45 amino acids including six toeight conserved cysteine residues. Defensins are classified in threegroups, α-defensins, β-defensins and θ-defensins.

In another embodiment, the TF region further contains a repeat sequenceincluding, but not limited to, (PVDPRLEPWKHPGSQP)_(n) (SEQ ID NO:12)wherein n=2-10 at the N-terminus. Furthermore, the repeat sequence canbe separated from the N-terminus of an immunosuppressive Tat derivativepolypeptide by one or more amino acids acting as a spacer.

In another embodiment, the amino acid sequence of the TF region is oneof SEQ ID NOs:36, 39, 44, 48, 50, 54, 59, 60, or 61. In anotherembodiment, the amino acid sequence of the cysteine-rich region is oneof SEQ ID NOs:37, 40, 41, 43, 45, 47, 49, 51, 53, 55, 57, 58, 62, 63, 64or 70. In another embodiment, the amino acid sequence of the C-terminalregion is one of SEQ ID NOs:38, 42, 46, 47, 49, 52, 53, 68 or 71. In yetanother embodiment, both the cysteine-rich region and the C-terminalregion are from the same source and the amino acid sequence of thecysteine-rich/C-terminal region is one of SEQ ID NO:47, 49, or 53.

In additional embodiments, one or more amino acids, including but notlimited to proline, in the TF domain is deleted or substituted with aconservative amino acid substitution.

In additional embodiments the use of conservatively modified variants ofthe immunosuppressive Tat derivatives is provided. The variantsdescribed herein maintain the biological activity of the parent orsource molecule.

TABLE 1 Source† (SEQ ID NO:) cysteine-rich C-terminal SEQ ID NO.Amino Acid Sequence TF region region region 6MEPVDANLEAWKHAGSQPRKTACTTCYCKKCCFHCQVCFTRKGL HIV-1 HIV-1 H IV-1(Nani-P1) GISYGRKKRRQRRRAPQDSQTHQASLSKQPASQSRGDPTGPTES SEQ ID NO: 59SEQ ID NO: 62 SEQ ID NO: 42 KKKVERETETDPFD 7MDPKGEEDQDVSHQDLIKQYRKPRTACNNCYCKKCCFHCYACFL SIVagm^(a) HIV-1 SIVagm(Nani-P2) RKGLGITYHAFRTRRKKIASADRIPVPQQSISIRGRDSQTTQESQK SEQ ID NO: 60SEQ ID NO: 63 SEQ ID NO: 68 KVEEQAKANLRISRKNLGDETRGPVGAGN 8METPLKEQENSLESCREHSSSISEVDVPTPVSCLRKGGRCWNRCI SIVsmm^(b) Murine β- HIV-1(Nani-P3) GNTRQIGSCGVPFLKCCKRKPFTRKGLGISYGRKKRRQRRRAPQ SEQ ID NO: 61defensin-3 SEQ ID NO: 42 DSQTHQASLSKQPASQSRGDPTGPTESKKKVERETETDPFDSEQ ID NO: 64 9 MDPIDPDLEPWKHPGSQPETACNNCFCKKCSYHCLVCFQKKGLG SIVcpz^(c)HIV-1 HIV-1 ISHGRKKRRQRRSAPPSSEDHQNLISKQPIPRTQGDQTGSEESKK SEQ ID NO: 39SEQ ID NO: 65 SEQ ID NO: 42 KVESKTETDPFD 10MEPLTPHPWVYSGGQPKVPTTACSKCYCKICCWHCQLCLKKGLG Human hr gene HIV-1 HIV-1ISYGRKKRRQRRRAPQDSQTHQASLSKQPASQSRGDPTGPTESK SEQ ID NO: 48 SEQ ID NO: 66SEQ ID NO: 42 KKVERETETDPFD 11MAGPHPVIVITGPHEEPRKTACTTCYCKKCCFHCQVCFTRKGLGIS VIVIT^(d) HIV-1 HIV-1YGRKKRRQRRRAPQDSQTHQASLSKQPASQSRGDPTGPTESKK SEQ ID NO: 50 SEQ ID NO: 67SEQ ID NO: 42 KVERETETDPFD 13MDPTDPELPPWQQPGSQPPTPRKTACTTCYCKKCCFHCQVCFL SIVgor^(e) HIV-1 SIVsyk^(f)QKGLGITYARPRKRAARSISEDDSAPTEPYPGPEGPRQTRRRRRR SEQ ID NO: 36SEQ ID NO: 37 SEQ ID NO: 38 QWRQRRTQRLYLQQRIFEAIFGSRTAALEDSLQQLQISD 14MDPIDPDLEPWKHPGSQPETACNNCFCKKCSYHCLVCFLQKGLGI SIVcpz HIV-1 SIVsykTYARPRKRAARSISEDDSAPTEPYPGPEGPRQTRRRRRRQWRQ SEQ ID NO: 39 SEQ ID NO: 40SEQ ID NO: 38 RRTQRLYLQQRIFEAIFGSRTAALEDSLQQLQISD 15MDPIDPDLEPWKHPGSQPACYCRIPACIAGERRYGTCIYQGRLWA SIVcpz Human α- HIV-1FCCFHCQVCFTRKGLGISYGRKKRRQRRRAPQDSQTHQASLSKQ SEQ ID NO: 39 defensin-1SEQ ID NO: 42 PASQSRGDPTGPTESKKKVERETETDPFD SEQ ID NO: 41 16MDPIDPDLEPWKHPGSQPGIGDPVTCLKSGAICHPVFCPRRYKQI SIVcpz Human β- HIV-1GTCGLPGTKCCKKPFHCQVCFTRKGLGISYGRKKRRQRRRAPQD SEQ ID NO: 39 defensin-2SEQ ID NO: 42 SQTHQASLSKQPASQSRGDPTGPTESKKKVERETETDPFD SEQ ID NO: 43 17MEPVDPRLEPWKHPGSQPKTACNNCHCKVCCYHCVYCFTKKGL HIV-1 SIVcpz SIVgorGISYGRKKRRRPARTADKDQDNQDPVSKQSLAGTRSQQE SEQ ID NO: 44 SEQ ID NO: 45SEQ ID NO: 46 18 MDPIDPDLEPWKHPGSQPTTACSKCYCKICCWHCQLCLKKGLGIS SIVcpzHIV-1 YGRKKRKHRRGTPQSSKDHQNPIPEQPLPIIRGNPTDPKESKKEV SEQ ID NO: 39SEQ ID NO: 47 ASKAETDPFD 19 MEPLTPHPWVYSGGQPKVPETACNNCFCKKCSYHCLVCFQKKGLHuman hr gene HIV-1 GISHGRKKRRQRRSAPPSSEDHQNLISKQPIPRTQGDQTGSEESKSEQ ID NO: 48 SEQ ID NO: 49 KKVESKTETDPFD 20MAGPHPVIVITGPHEEPTTACSKCYCKICCWHCQLCLKKGLGISYG VIVIT HIV-1RKKRKHRRGTPQSSKDHQNPIPEQPLPIIRGNPTDPKESKKEVAS SEQ ID NO: 50SEQ ID NO: 47 KAETDPFD 21 MAGPHPVIVITGPHEEPETACNNCFCKKCSYHCLVCFQKKGLGISVIVIT HIV-1 HGRKKRRQRRSAPPSSEDHQNLISKQPIPRTQGDQTGSEESKKK SEQ ID NO: 50SEQ ID NO: 49 VESKTETDPFD 22MEPLTPHPWVYSGGQPKVPRTCHCRSRCLRRESNSGSCNINGRI Human hr gene Myeloid α-SIVmac^(h) SSLCCFLKKGLGISYEKSHRRRRTPKKAKANTSSASNEPIPNRIRL SEQ ID NO: 48defensin-9^(g) SEQ ID NO: 52 CQPKKAKKETVEAAVATAPGLGR SEQ ID NO: 51 23MAGPHPVIVITGPHEEPRTCHCRSRCLRRESNSGSCNINGRISSLC VIVIT Myeloid α- SIVmacCFLKKGLGISYEKSHRRRRTPKKAKANTSSASNEPIPNRIRLCQPK SEQ ID NO: 50 defensin-9SEQ ID NO: 52 KAKKETVEAAVATAPGLGR SEQ ID NO: 51 24MDPIDPDLEPWKHPGSQPRTCHCRSRCLRRESNSGSCNINGRIS SIVcpz Myeloid α- SIVmacSLCCFLKKGLGISYEKSHRRRRTPKKAKANTSSASNEPIPNRIRLC SEQ ID NO: 39 defensin-9SEQ ID NO: 52 QPKKAKKETVEAAVATAPGLGR SEQ ID NO: 51 25MEPLTPHPWVYSGGQPKVPLEACYNKCYCKRCCYHCQHCFLKK Human hr gene SIVsmmGLGICYEQQRRRTPKKTKANTSSASDKSLSRRARNCQPKKEKKET SEQ ID NO: 48SEQ ID NO: 53 VEAEVATDLGLGR 26MAGPHPVIVITGPHEEPLEACYNKCYCKRCCYHCQHCFLKKGLGI VIVIT SIVsmmCYEQQRRRTPKKTKANTSSASDKSLSRRARNCQPKKEKKETVEA SEQ ID NO: 50 SEQ ID NO: 53EVATDLGLGR 27 MDPIDPDLEPWKHPGSQPLEACYNKCYCKRCCYHCQHCFLKKGL SIVcpz SIVsmmGICYEQQRRRTPKKTKANTSSASDKSLSRRARNCQPKKEKKETVE SEQ ID NO: 39SEQ ID NO: 53 AEVATDLGLGR 28 MMEPVDPDLPKEQHPPATPRCESCKLGRGRCRKECLENEKPDGSIVmon^(i) β-defensin-105^(j) HIV-1RCRLNFLCCFHCQVCFTRKGLGISYGRKKRRQRRRAPQDSQTHQ SEQ ID NO: 54 SEQ ID NO: 55SEQ ID NO: 42 ASLSKQPASQSRGDPTGPTESKKKVERETETDPFD 29MEPLTPHPWVYSGGQPKVPCESCKLGRGRCRKECLENEKPDGR Human hr gene β-defensin-105HIV-1 CRLNFLCCFHCQVCFTRKGLGISYGRKKRRQRRRAPQDSQTHQA SEQ ID NO: 48SEQ ID NO: 55 SEQ ID NO: 42 SLSKQPASQSRGDPTGPTESKKKVERETETDPFD 30MDPIDPDLEPWKHPGSQPCESCKLGRGRCRKECLENEKPDGRC SIVcpz β-defensin-105 HIV-1RLNFLCCFHCQVCFTRKGLGISYGRKKRRQRRRAPQDSQTHQAS SEQ ID NO: SEQ ID NO: 55SEQ ID NO: 42 LSKQPASQSRGDPTGPTESKKKVERETETDPFD 31MAGPHPVIVITGPHEEPCESCKLGRGRCRKECLENEKPDGRCRLN VIVIT β-defensin-105 HIV-1FLCCFHCQVCFTRKGLGISYGRKKRRQRRRAPQDSQTHQASLSK SEQ ID NO: 50 SEQ ID NO: 55SEQ ID NO: 42 QPASQSRGDPTGPTESKKKVERETETDPFD 32MEPVDPRLEPWKHPGSQPPEPVDPRLEPWKHPGSQPKTACNNC HIV-1 SIVcpz HIV-1HCKVCCYHCVYCFFHCQVCFTRKGLGISYGRKKRRQRRRAPQDS SEQ ID NO: 56 SEQ ID NO: 57SEQ ID NO: 42 QTHQASLSKQPASQSRGDPTGPTESKKKVERETETDPFD 33MEPLTPHPWVYSGGQPKVPLRCICRRGICRLLQRRYGSCAFPGR Human hr gene⊖-defensin^(k) SIVsmm LYRICCFLKKGLGICYEQQRRRTPKKTKANTSSASDKSLSRRARNSEQ ID NO: 48 SEQ ID NO: 58 SEQ ID NO: 52 CQPKKEKKETVEAEVATDLGLGR 34MAGPHPVIVITGPHEEPLRCICRRGICRLLQRRYGSCAFPGRLYRI VIVIT ⊖-defensin SIVsmmCCFLKKGLGICYEQQRRRTPKKTKANTSSASDKSLSRRARNCQPK SEQ ID NO: 50SEQ ID NO: 58 SEQ ID NO: 52 KEKKETVEAEVATDLGLGR 35MDPIDPDLEPWKHPGSQPLRCICRRGICRLLQRRYGSCAFPGRLY SIVcpz ⊖-defensin SIVsmmRICCFLKKGLGICYEQQRRRTPKKTKANTSSASDKSLSRRARNCQ SEQ ID NO: 39SEQ ID NO: 58 SEQ ID NO: 52 PKKEKKETVEAEVATDLGLGR 69MEPLTPHPVVVYSGGQPKVPLEACYNKCYCKRCCYHCQHCFSKK Human hr gene SIVsmm HIV-2GLGISYERKGRRRRTPRKTKTPSPSAPDKSISTRTGDSQPTKEQK SEQ ID NO: 48SEQ ID NO: 70 SEQ ID NO: 71 KTSEATVVTTCGLGQ ^(a)agm = African GreenMonkey; ^(b)SMM = sooty mangabey monkey; ^(c)cpz = chimpanzee; ^(d)VIVIT= artificial TF sequence; ^(e)gor = gorilla; ^(f)syk = Sykes monkey;^(g)from Macaca mulatta; ^(h)mac = macaque; imon = Mona monkey; ^(j)fromChlorocebus aethiops; ^(k)from Pongo abelli.

As used herein the term “conservatively modified variants” refers tovariant peptides which have the same or similar biological activity ofthe original peptides. For example, conservative amino acid changes maybe made, which although they alter the primary sequence of the proteinor peptide, do not alter its function. A conservative variant has atleast one amino acid substituted by another amino acid or an amino acidanalog that has at least one property similar to that of the originalamino acid from an exemplary reference peptide. Examples of propertiesinclude, without limitation, similar size, topography, charge,hydrophobicity, hydrophilicity, lipophilicity, covalent-bondingcapacity, hydrogen-bonding capacity, a physicochemical property, of thelike, or any combination thereof. A conservative substitution can beassessed by a variety of factors, such as, e.g., the physical propertiesof the amino acid being substituted (Table 2) or how the original aminoacid would tolerate a substitution (Table 3). The selections of whichamino acid can be substituted for another amino acid in a peptidedisclosed herein are known to a person of ordinary skill in the art. Aconservative variant can function in substantially the same manner asthe exemplary reference peptide, and can be substituted for theexemplary reference peptide in any aspect of the present specification.

TABLE 2 Amino Acid Properties Property Amino Acids Aliphatic G, A, I, L,M, P, V Aromatic F, H, W, Y C-beta branched I, V, T Hydrophobic C, F, I,L, M, V, W Small polar D, N, P Small non-polar A, C, G, S, T Large polarE, H, K, Q, R, W, Y Large non-polar F, I, L, M, V Charged D, E, H, K, RUncharged C, S, T Negative D, E Positive H, K, R Acidic D, E Basic K, RAmide N, Q

TABLE 3 Amino Acid Substitutions Amino Favored Acid Substitution NeutralSubstitutions Disfavored substitution A G, S, T C, E, I, K, M, L, P, Q,R, V D, F, H, N, Y, W C F, S, Y, W A, H, I, M, L, T, V D, E, G, K, N, P,Q, R D E, N G, H, K, P, Q, R, S, T A, C, I, L, E D, K, Q A, H, N, P, R,S, T C, F, G, I, L, M, V, W, Y F M, L, W, Y C, I, V A, D, E, G, H, K, N,P, Q, R, S, T G A, S D, K, N, P, Q, R C, E, F, H, I, L, M, T, V, W, Y HN, Y C, D, E, K, Q, R, S, T, W A, F, G, I, L, M, P, V I V, L, M A, C, T,F, Y D, E, G, H, K, N, P, Q, R, S, W K Q, E, R A, D, G, H, M, N, P, S, TC, F, I, L, V, W, Y L F, I, M, V A, C, W, Y D, E, G, H, K, N, P, Q, R,S, T M F, I, L, V A, C, R, Q, K, T, W, Y D, E, G, H, N, P, S N D, H, SE, G, K, Q, R, T A, C, F, I, L, M, P, V, W, Y P — A, D, E, G, K, Q, R,S, T C, F, H, I, L, M, N, V, W, Y Q E, K, R A, D, G, H, M, N, P, S, T C,F, I, L, V, W, Y R K, Q A, D, E, G, H, M, N, P, S, T C, F, I, L, V, W, YS A, N, T C, D, E, G, H, K, P, Q, R, T F, I, L, M, V, W, Y T S A, C, D,E, H, I, K, M, N, P, F, G, L, W, Y Q, R, V V I, L, M A, C, F, T, Y D, E,G, H, K, N, P, Q, R, S, W W F, Y H, L, M A, C, D, E, G, I, K, N, P, Q,R, S, T, V Y F, H, W C, I, L, M, V A, D, E, G, K, N, P, Q, R, S, TMatthew J. Betts and Robert, B. Russel, Amino Acid Properties andConsequences of Substitutions, pp. 289-316, In Bioinformatics forGeneticists, (eds Michael R. Barnes, Ian C. Gray, Wiley, 2003).

TABLE 4 SIV strain abbreviations useful in Tat derivative peptides SIVhost designation SIV Host Species Latin designation SIVagmVer (AfricanGreen Monkey) Vervet Chlorocebus pygetythrus SIVagmGri (African GreenMonkey) Grivet Chlorocebus aethiops SIVagmTan (African Green Monkey)Tantalus Chlorocebus tantalus SIVagmSab (African Green Monkey) SabeusChlorocebus sabaeus SIVrcm Red-capped Mangabey Cercocebus torquatustorquatus SIVsyk Sykes Monkey Cercopithecus albogularis SIVagi AgileMangabey Cercocebus agilis SIVsun Sun-tailed Monkey Cercopithecussolatus SIVIho L'Hoests Monkey Cercopithecus lhoesti SIVstm Stump-tailMacaque Macaca arctoides SIVmac Macaque Macaca mulatta SIVsmm Sootymangabey monkey Cercocebus atys atys SIVmnd Mandrill Mandrillus sphinxSIVdr1 Drill Monkey Mandrillus leucophaeus SIVtal Talapoin MonkeyMiopithecus talapoin SIVmus Mustached Monkey Cercopithecus cephus SIVdebDe Brazza's Monkey Cercopithecus neglectus SIVden Dent's MonkeyCercopithecus denti SIVmon Mona Monkey Cercopithecus mona SIVgor GorillaGorilla gorilla SIVwrc Western Red Colobus Procolobus verus SIVcpzPttPan Troglodytes Troglodytes Pan troglodytes troglodytes SIVcpzPts PanTroglodytes Schweinfurthi Pan troglodytes schweinfurthii SIVmne Pig-tailMacaque Macaca nemestrina SIVasc Red-tailed Guenon Cercopithecusascanius schmidti SIVbab Yellow Baboon Papio spp. SIVbIc Bioko BlackColobus Monkey Cercopithecus satanas satanas SIVbkm Black MangabeyLophocebus aterrimus SIVblu Blue Monkey Cercopithecus mitis SIVcolColobus Monkey Colobus guereza SIVolc Oilve Colobus Monkey procolobusverus SIVgsn Greater Spot-nosed Monkey Cercopithecus nictitans SIVkrcKibale Red Colobus Moneky Procolobus [Piliocolobus] rufomitratustephrosceles SIVpat Patas Monkey Erythrocebus patas SIVpre PreussisMonkey Cercopithecus preussi SIVreg Red-eared Guenon Cercopithecuserythrotis etythrotis SIVtrc Tshuapa Red Colobus Piliocolobus tholloniSIVwcm White-crowned Mangabey Cercocebus torquatus lunulatus SIVwolWolf's Monkey Cercopithecus woffi

In one embodiment, an immunosuppressive Tat derivative polypeptide is apeptide disclosed in Table 1. An immunosuppressive Tat derivativepolypeptide can also comprise conservative variants to a Tat derivativepolypeptide. In an embodiment, a conservative variant of animmunosuppressive Tat derivative polypeptide is a conservative variantof an immunosuppressive Tat derivative polypeptide disclosed herein. Inaspects of this embodiment, a conservative variant of animmunosuppressive Tat derivative polypeptide can be, for example, anamino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 97%, or atleast 98%, or at least 99% amino acid sequence identity to animmunosuppressive Tat derivative polypeptide. In other aspects of thisembodiment, a conservative variant of an immunosuppressive Tatderivative polypeptide can be, for example, an amino acid sequencehaving at most 50%, 55%, 60%, 65%, 70%, 75%, at most 80%, at most 85%,at most 90%, at most 95%, at most 97%, or at most 98%, or at most 99%amino acid sequence identity to an immunosuppressive Tat derivativepolypeptide.

In other aspects of this embodiment, a conservative variant of animmunosuppressive Tat derivative polypeptide can be, for example, animmunosuppressive Tat derivative polypeptide having 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30 or more conservativesubstitutions, to the amino acid sequence of an immunosuppressive Tatderivative polypeptide. In other aspects of this embodiment, aconservative variant of an immunosuppressive Tat derivative polypeptidecan be, for example, an amino acid sequence having at least 1, at least2, at least 3, at least 4, at least 5, at least 6, at least 7, at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 20, or at least 25 conservativesubstitutions to the amino acid sequence of an immunosuppressive Tatderivative polypeptide. In yet other aspects of this embodiment, aconservative variant of an immunosuppressive Tat derivative polypeptidecan be, for example, an amino acid sequence having at most 1, at most 2,at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, atmost 9, at most 10, at most 11, at most 12, at most 13, at most 14, atmost 15, at most 20, at most 25, or at most 30 conservativesubstitutions to the amino acid sequence of an immunosuppressive Tatderivative polypeptide.

Modifications (which do not normally alter primary sequence) include invivo, or in vitro, chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g. by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

Also included are immunosuppressive Tat derivative polypeptides whichhave been modified using ordinary molecular biological techniques so asto improve their resistance to proteolytic degradation or to optimizesolubility properties. Analogs of such polypeptides include thosecontaining residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring synthetic amino acids. Thepeptides disclosed herein are not limited to products of any of thespecific exemplary processes listed herein.

In addition to substantially full length polypeptides, the presentdisclosure also provides for biologically active fragments of theimmunosuppressive Tat derivative polypeptides.

As used herein, amino acid sequences which are substantially the sametypically share more than 95% amino acid identity. It is recognized,however, that proteins (and DNA or mRNA encoding such proteins)containing less than the above-described level of identity arising assplice variants or that are modified by conservative amino acidsubstitutions (or substitution of degenerate codons) are contemplated tobe within the scope of the present disclosure. As readily recognized bythose of skill in the art, various ways have been devised to alignsequences for comparison, e.g., Blosum 62 scoring matrix, as describedby Henikoff and Henikoff in Proc. Natl. Acad Sci. USA 89:10915 (1992).Algorithms conveniently employed for this purpose are widely available(see, for example, Needleman and Wunsch in J. Mol. Bio. 48:443 (1970).

Therefore, disclosed herein are amino acid sequences 85%, 90%, 95%, 98%,99% or 100% identical to the immunosuppressive Tat derivatives disclosedin Table 1.

An immunosuppressive Tat derivative comprises an altered Tat peptide. Inan aspect of this embodiment, an immunosuppressive Tat derivativecomprises a Tat peptide in which amino acids 3-19 are altered. Inanother aspect of this embodiment, an immunosuppressive Tat derivativecomprises a Tat peptide in which amino acids 3-19 of SEQ ID NOs:6-11,13-35, or 69 are altered.

In other aspects of this embodiment, an immunosuppressive Tat derivativehas, e.g., at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 93%, at least 95%, at least 97%, or at least 99%amino acid identity with SEQ ID NOs: 6-11, 13-35, or 69. In yet otheraspects of this embodiment, an immunosuppressive Tat derivative has,e.g., at most 70%, at most 75%, at most 80%, at most 85%, at most 90%,at most 93%, at most 95%, at most 97%, or at most 99% amino acididentity with SEQ ID NOs: 6-11, 13-35, or 69.

In other aspects of this embodiment, an immunosuppressive Tat derivativehas, e.g., at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nineor at least ten contiguous amino acid substitutions, deletions, and/oradditions relative to SEQ ID NOs:6-11, 13-35, or 69. In yet otheraspects of this embodiment, an immunosuppressive Tat derivative has,e.g., at most one, at most two, at most three, at most four, at mostfive, at most six, at most seven, at most eight, at most nine or at mostten contiguous amino acid substitutions, deletions, and/or additionsrelative to SEQ ID NOs:6-11, 13-35, or 69.

In other aspects of this embodiment, an immunosuppressive Tat derivativehas, e.g., at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nineor at least ten non-contiguous amino acid substitutions, deletions,and/or additions relative to SEQ ID NOs:6-11, 13-35, or 69. In yet otheraspects of this embodiment, an immunosuppressive Tat derivative has,e.g., at most one, at most two, at most three, at most four, at mostfive, at most six, at most seven, at most eight, at most nine or at mostten non-contiguous amino acid substitutions, deletions, and/or additionsrelative to SEQ ID NOs:6-11, 13-35, or 69.

Furthermore, the peptides disclosed herein can self-associate intomultimers, including but not limited to, dimers, trimers, and tetramers,in addition to existing in the monomer form. Multimerization of peptidescan occur spontaneously or can be facilitated by subjecting the peptidesto conditions conducive to multimerization. These conditions are knownto persons of ordinary skill in peptide chemistry. The compositionsdisclosed herein can include monomers or multimers of the peptides, or amixture of monomers and multimers.

The following expression systems are suitable for use in expressing thedisclosed immunosuppressive Tat derivatives: mammalian cell expressionsystems such as, but not limited to, insect cell expression systems suchas, but not limited to, Bac-to-Bac expression system, baculovirusexpression system, and DES expression systems; and E. coli expressionsystems including, but not limited to, pET, pSUMO and GST expressionsystems. In another embodiment, the Tat derivatives are expressed with ahistidine tag useful for isolation of the polypeptide. Histidine tagpurification systems are known to persons of ordinary skill in the art.Furthermore, the immunosuppressive Tat derivatives can be synthesized byconventional chemical synthesis techniques known to persons of ordinaryskill in the art.

As used herein, the terms “treating” or “treatment” when used inreference to a disorder include preventing at least one symptom of thecondition, i.e., causing a clinical symptom to not significantly developin a subject that may be exposed to, or predisposed to, the disease butdoes not yet experience or display symptoms of the disease, inhibitingthe disease, i.e., arresting or reducing the development of the diseaseor its symptoms, or relieving the disease, i.e., causing regression ofthe disease or its clinical symptoms. Treatment, prevention, andameliorating a condition, as used herein, can include, for exampledecreasing or eradicating a deleterious or harmful condition associatedwith inflammation, an autoimmune disorder, or a neurodegenerativedisease.

“Therapeutically effective amount” is intended to qualify the amountrequired to achieve a therapeutic effect.

The present disclosure is also directed to pharmaceutical compositionscomprising the above-described immunosuppressive Tat derivativepolypeptides. Dosages and desired drug concentrations of the disclosedpharmaceutical compositions may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mardenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al, Eds., PergamonPress, New York 1989, pp. 42-96. In one embodiment, the disease ispresent. In another embodiment, the life of a cell or an individual isprolonged due to the methods described herein.

Accordingly, the compositions designed for oral, nasal, lingual,sublingual, buccal, intrabuccal, intravenous, subcutaneous,intramuscular and pulmonary administration can be made without undueexperimentation by means well known in the art, for example with aninert diluent or with an pharmaceutically acceptable carrier. For thepurpose of therapeutic administration, the pharmaceutical compositionsmay be incorporated with excipients and used in the form of tablets,troches, capsules, elixirs, suspensions, solutions, syrups, and thelike. A “pharmaceutically acceptable carrier” means any of the standardpharmaceutical carriers. Examples of suitable carriers are well known inthe art and may include but are not limited to any of the standardpharmaceutical carriers like phosphate buffered saline solutions,phosphate buffered saline containing Polysorb 80, water, emulsions suchas oil/water emulsion, and various types of wetting agents. Othercarriers may also include sterile solutions, tablets, coated tablets,and capsules. Typically such carriers contain excipients like starch,milk, sugar, certain types of clay, gelatin, stearic acid or saltsthereof, magnesium or calcium stearate, talc, vegetable fats or oils,gums, glycols, or other known excipients. Compositions comprising suchcarriers are formulated by well known conventional methods.

The immunosuppressive Tat derivative polypeptide compositions can easilybe administered parenterally such as for example, by intravenous,intramuscular, intrathecal or subcutaneous injection. Parenteraladministration can be accomplished by incorporating the compounds into asolution or suspension. Such solutions or suspensions may also includesterile diluents such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents. Parenteral formulations may also includeantibacterial agents such as for example, benzyl alcohol or methylparabens, antioxidants such as for example, ascorbic acid or sodiumbisulfite and chelating agents such as EDTA. Buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose may also be added. The parenteralpreparation can be enclosed in ampules, disposable syringes or multipledose vials made of glass or plastic.

Transdermal administration includes percutaneous absorption of thecomposition through the skin. Transdermal formulations include patches,iontophoresis devices, ointments, creams, gels, salves and the like.

The composition may include various materials which modify the physicalform of a solid or liquid dosage unit. For example, the composition mayinclude materials that form a coating shell around the activeingredients. The materials which form the coating shell are typicallyinert, and may be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients may beencased in a gelatin capsule or cachet.

The immunosuppressive Tat derivative polypeptide compositions of thepresent disclosure may be administered in a therapeutically effectiveamount, according to an appropriate dosing regimen. As understood by askilled artisan, the exact amount required may vary from subject tosubject, depending on the subject's species, age and general condition,the severity of the disease, the particular agent(s) and the mode ofadministration. In some embodiments, about 0.0001 mg/kg to about 50mg/kg, of the composition based on the subject's body weight isadministered, one or more times a day, to obtain the desired therapeuticeffect. In other embodiments, about 0.001 mg/kg to about 50 mg/kg, about0.01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 50 mg/kg, about 1mg/kg to about 50 mg/kg, about 10 mg/kg to about 50 mg/kg, about 0.0001mg/kg to about 25 mg/kg, about 0.0001 mg/kg to about 10 mg/kg, about0.0001 mg/kg to about 5 mg/kg, about 0.0001 mg/kg to about 1 mg/kg,about 1 mg/kg to about 45 mg/kg, about 1 mg/kg to about 40 mg/kg, about1 mg/kg to about 35 mg/kg, about 1 mg/kg to about 30 mg/kg, about 1mg/kg to about 25 mg/kg, about 1 mg/kg to about 20 mg/kg, about 1 mg/kgto about 15 mg/kg, about 1 mg/kg to about 10 mg/kg, or about 1 mg/kg toabout 5 mg/kg of the composition based on the subject's body weight isadministered, one or more times a day, to obtain the desired therapeuticeffect.

The total daily dosage of the compositions will be determined by theattending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientor subject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; the activity of thespecific compound employed; the specific composition employed; the age,body weight, general health, sex and diet of the patient or subject; thetime of administration, route of administration, and rate of excretionof the specific compound employed; the duration of the treatment; drugsused in combination or coincidental with the specific compound employed,and other factors well known in the medical arts.

The disclosed compositions may also be employed in combinationtherapies. That is, the compositions presently disclosed can beadministered concurrently with, prior to, or subsequent to, one or moreother desired compositions, therapeutics, treatments or medicalprocedures. The particular combination of therapies administered will bedetermined by the attending physician and will take into accountcompatibility of the treatments and the desired therapeutic effect to beachieved. It will be appreciated that therapeutically active agentsutilized in combination may be administered together in a singlecomposition, treatment or procedure, or alternatively may beadministered separately. Suitable therapeutic agents include, but arenot limited to, immunosuppressive agents, anti-inflammatory agents,chemotherapeutic agents, immunomodulatory agents, biologic agents, andsmall molecules.

In another embodiment, repetitive, or frequent, dosing of the disclosedimmunosuppressive Tat derivatives is contemplated. Frequent dosing isone procedure used for example in allergy therapy that can supportimmunological tolerance to an agent.

The number of repeated doses of the immunosuppressive Tat derivativescan be established by the medical professional based on the response ofthe patient to the doses. In one embodiment, the immunosuppressive Tatderivative is administered once every three days for 3 doses in a tenday period. This administration scheme is then repeated for a pluralityof cycles. The present disclosure envisions a variety of differentadministration schemes wherein the immunosuppressive Tat derivative isadministered multiple times within a selected time frame and then theadministration scheme is repeated for a plurality of cycles. In anotherembodiment, administration of the immunosuppressive Tat derivative canbe alternated with administration of one or more other therapeuticagents.

Aspects of the present specification provide, in part, a compositioncomprising an immunosuppressive Tat derivative polypeptide. Animmunosuppressive Tat derivative polypeptide includes the compoundsdisclosed herein. The compositions disclosed herein may, or may not,comprise any number and combination of compounds disclosed herein. Forinstance, a composition can comprise two or more compounds disclosedherein or three or more compounds disclosed herein.

A compound disclosed herein, or a composition comprising such acompound, is generally administered to an individual as a pharmaceuticalcomposition. Pharmaceutical compositions may be prepared by combining atherapeutically effective amount of at least one compound as disclosedherein as an active ingredient, with conventional acceptablepharmaceutical excipients, and by preparation of unit dosage formssuitable for therapeutic use. As used herein, the term “pharmaceuticalcomposition” and refers to a therapeutically effective concentration ofan active compound, such as, e.g., any of the compounds disclosedherein. Preferably, the pharmaceutical composition does not produce anadverse, allergic, or other untoward or unwanted reaction whenadministered to an individual. A pharmaceutical composition disclosedherein is useful for medical and veterinary applications. Apharmaceutical composition may be administered to an individual alone,or in combination with other supplementary active compounds, agents,drugs or hormones. The pharmaceutical compositions may be manufacturedusing any of a variety of processes, including, without limitation,conventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, and lyophilizing. Thepharmaceutical composition can take any of a variety of forms including,without limitation, a sterile solution, suspension, emulsion,lyophilizate, powder, syrup, elixir, or any other dosage form suitablefor administration.

Liquid dosage forms suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethyleneglycol (PEG), glycerol, and the like), suitable mixturesthereof, vegetable oils (such as olive oil) and injectable organicesters such as ethyl oleate. Proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersions and by the use ofsurfactants. In liquid formulations, a therapeutically effective amountof a compound disclosed herein may be between about 0.0001% (w/v) toabout 50% (w/v), about 0.001% (w/v) to about 10.0% (w/v), or about 0.01%(w/v) to about 1.0% (w/v).

A pharmaceutical composition disclosed herein can optionally include apharmaceutically acceptable carrier that facilitates processing of anactive compound into pharmaceutically acceptable compositions. As usedherein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem complicationscommensurate with a reasonable benefit/risk ratio. As used herein, theterm “pharmacologically acceptable carrier” is synonymous with“pharmacological carrier” and refers to any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, additive, auxiliary, or excipient.” Such acarrier generally is mixed with an active compound or permitted todilute or enclose the active compound and can be a solid, semi-solid, orliquid agent. It is understood that the active compounds can be solubleor can be delivered as a suspension in the desired carrier or diluent.Any of a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., water,saline, glycine, hyaluronic acid and the like; solid carriers such as,e.g., starch, magnesium stearate, mannitol, sodium saccharin, talcum,cellulose, glucose, sucrose, lactose, trehalose, magnesium carbonate,and the like; solvents; dispersion media; coatings; antibacterial andantifungal agents; isotonic and absorption delaying agents; or any otherinactive ingredient. Selection of a pharmacologically acceptable carriercan depend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activecompound, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in Pharmaceutical Dosage Forms andDrug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams& Wilkins Publishers, 7^(th) ed. 1999); Remington: The Science andPractice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams &Wilkins, 20^(th) ed. 2000); Goodman & Gilman's The Pharmacological Basisof Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill Professional,10^(th) ed. 2001); and Handbook of Pharmaceutical Excipients (Raymond C.Rowe et al., APhA Publications, 4^(th) edition 2003). These protocolsare routine and any modifications are well within the scope of oneskilled in the art and from the teaching herein.

A pharmaceutical composition disclosed herein can optionally include,without limitation, other pharmaceutically acceptable components (orpharmaceutical components), including, without limitation, buffers,preservatives, tonicity adjusters, salts, antioxidants, osmolalityadjusting agents, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed herein,provided that the resulting preparation is pharmaceutically acceptable.Such buffers include, without limitation, acetate buffers, boratebuffers, citrate buffers, phosphate buffers, neutral buffered saline,and phosphate buffered saline. It is understood that acids or bases canbe used to adjust the pH of a composition as needed. Pharmaceuticallyacceptable antioxidants include, without limitation, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole, and butylated hydroxytoluene. Useful preservativesinclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilizedoxy chloro composition, such as, e.g., sodium chlorite and chelants,such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, andCaNaDTPA-bisamide. Tonicity adjustors useful in a pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor.

A compound disclosed herein, or a composition comprising such acompound, may also be incorporated into a drug delivery platform inorder to achieve a controlled compound release profile over time. Such adrug delivery platform comprises a compound disclosed herein dispersedwithin a polymer matrix, typically a biodegradable, bioerodible, and/orbioresorbable polymer matrix. As used herein, the term “polymer” refersto synthetic homo- or copolymers, naturally occurring homo- orcopolymers, as well as synthetic modifications or derivatives thereofhaving a linear, branched or star structure. Copolymers can be arrangedin any form, such as, e.g., random, block, segmented, tapered blocks,graft, or triblock. Polymers are generally condensation polymers.Polymers can be further modified to enhance their mechanical ordegradation properties by introducing cross-linking agents or changingthe hydrophobicity of the side residues. If crosslinked, polymers areusually less than 5% crosslinked, usually less than 1% crosslinked.

Suitable polymers include, without limitation, alginates, aliphaticpolyesters, polyalkylene oxalates, polyamides, polyamidoesters,polyanhydrides, polycarbonates, polyesters, polyethylene glycol,polyhydroxyaliphatic carboxylic acids, polyorthoesters, polyoxaesters,polypeptides, polyphosphazenes, polysaccharides, and polyurethanes. Thepolymer usually comprises at least about 10% (w/w), at least about 20%(w/w), at least about 30% (w/w), at least about 40% (w/w), at leastabout 50% (w/w), at least about 60% (w/w), at least about 70% (w/w), atleast about 80% (w/w), or at least about 90% (w/w) of the drug deliveryplatform. Examples of biodegradable, bioerodible, and/or bioresorbablepolymers and methods useful to make a drug delivery platform aredescribed in, e.g., U.S. Pat. No. 4,756,911; U.S. Pat. No. 5,378,475;U.S. Pat. No. 7,048,946; U.S. Patent Publication 2005/0181017; U.S.Patent Publication 2005/0244464; U.S. Patent Publication 2011/0008437;each of which is incorporated by reference in its entirety.

In aspects of this embodiment, a polymer composing the matrix is apolypeptide such as, e.g., silk fibroin, keratin, or collagen. In otheraspects of this embodiment, a polymer composing the matrix is apolysaccharide such as, e.g., cellulose, agarose, elastin, chitosan,chitin, or a glycosaminoglycan like chondroitin sulfate, dermatansulfate, keratan sulfate, or hyaluronic acid. In yet other aspects ofthis embodiment, a polymer composing the matrix is a polyester such as,e.g., D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid,caprolactone, and combinations thereof.

One of ordinary skill in the art appreciates that the selection of asuitable polymer for forming a suitable disclosed drug delivery platformdepends on several factors. The more relevant factors in the selectionof the appropriate polymer(s), include, without limitation,compatibility of polymer with drug, desired release kinetics of drug,desired biodegradation kinetics of platform at implantation site,desired bioerodible kinetics of platform at implantation site, desiredbioresorbable kinetics of platform at implantation site, in vivomechanical performance of platform, processing temperatures,biocompatibility of platform, and patient tolerance. Other relevantfactors that, to some extent, dictate the in vitro and in vivo behaviorof the polymer include the chemical composition, spatial distribution ofthe constituents, the molecular weight of the polymer and the degree ofcrystallinity.

A drug delivery platform includes both a sustained release drug deliveryplatform and an extended release drug delivery platform. As used herein,the term “sustained release” refers to the release of a compounddisclosed herein over a period of about seven days or more. As usedherein, the term “extended release” refers to the release of a compounddisclosed herein over a period of time of less than about seven days.

In aspects of this embodiment, a sustained release drug deliveryplatform releases a compound disclosed herein with substantially firstorder release kinetics over a period of, e.g., about 7 days afteradministration, about 15 days after administration, about 30 days afteradministration, about 45 days after administration, about 60 days afteradministration, about 75 days after administration, or about 90 daysafter administration. In other aspects of this embodiment, a sustainedrelease drug delivery platform releases a compound disclosed herein withsubstantially first order release kinetics over a period of, e.g., atleast 7 days after administration, at least 15 days afteradministration, at least 30 days after administration, at least 45 daysafter administration, at least 60 days after administration, at least 75days after administration, or at least 90 days after administration.

In aspects of this embodiment, a drug delivery platform releases acompound disclosed herein with substantially first order releasekinetics over a period of, e.g., about 1 day after administration, about2 days after administration, about 3 days after administration, about 4days after administration, about 5 days after administration, or about 6days after administration. In other aspects of this embodiment, a drugdelivery platform releases a compound disclosed herein withsubstantially first order release kinetics over a period of, e.g., atmost 1 day after administration, at most 2 days after administration, atmost 3 days after administration, at most 4 days after administration,at most 5 days after administration, or at most 6 days afteradministration.

Aspects of the present disclosure provide, in part, an autoimmunedisorder. An autoimmune disorder arises from an overactive immuneresponse of the body against substances and tissues normally present inthe body resulting in a break in tolerance toward self-antigens. Inother words, the body actually attacks its own cells because the immunesystem mistakes some part of the body as a pathogen and attacks it.Characterized by the development of pathogenic T cell populationsinfiltrating the target organ or tissue, autoimmune disorders may berestricted to certain organs or involve a particular tissue in differentplaces.

Aspects of the present disclosure provide, in part, an inflammation.Inflammation refers to the actual tissue response (edema, erythema, etc)to a noxious stimulus. Neurogenic inflammation refers to the fact thatthis tissue response is initiated and/or maintained through the releaseof inflammatory mediators from peripheral sensory nerve terminals (i.e.,an efferent function, in contrast to the normal afferent signaling tothe spinal cord in these nerves). Neurogenic inflammation encompasses aseries of vascular and non-vascular inflammatory responses mediated by acomplex biological process that ultimately results in the local releaseof inflammatory mediators and sensitizing compounds from sensoryneurons. Upon insult by a noxious stimulus, such as, e.g., a pathogen,damage to cells, or an irritant, inflammation mediating and sensitizingmolecules, such as, e.g., histamine, prostaglandins, leukotrienes,serotonin, neutral proteases, cytokines, bradykinin and nitric oxide,are released from inflammation mediating cells, such as, e.g., mastcells, immune cells, vascular endothelial cells, and vascular smoothmuscle cells. See Richardson and Vasko, J. Pharmacol. Exp. Ther.302:839-845, (2002), which is hereby incorporated by reference in itsentirety. These inflammation mediating and sensitizing molecules act onsensory neurons to stimulate the release of inflammation inducingmolecules such as, e.g., neuropeptides like substance P (SP) andcalcitonin gene-related peptide (CGRP), prostaglandins, and amino acidslike glutamate, from the peripheral nerve endings. Upon release, theseinflammation inducing molecules are responsible for eliciting aninflammatory response, typically characterized by edema (swellingsecondary to plasma extravasation), hypersensitivity (secondary toalterations in the excitability of certain sensory neurons), and anerythema (redness and warmth secondary to vasodilation) which extendsbeyond the site of stimulation (the flare response). Id. Because theunderlying inflammatory symptoms are triggered by the activation ofprimary sensory neurons and the subsequent release of inflammationinducing molecules, the response is termed neurogenic inflammation.

Inflammation includes both acute inflammation and chronic inflammation.As used herein, the term “acute inflammation” means an inflammatoryresponse having pathophysiology effects where at least one of theunderlying symptoms being treated is due to a noxious stimulus etiology,such as, e.g., an antimicrobial response. As used herein, the term“chronic inflammation” means an inflammatory response havingpathophysiology effects where at least one of the underlying symptomsbeing treated is due to a nociceptive sensory nerve-based etiology, suchas, e.g., the release of an inflammation inducing molecule. Chronicinflammation includes both primary neurogenic inflammation and secondaryneurogenic inflammation. As used herein, the term “primary” neurogenicinflammation refers to tissue inflammation (inflammatory symptoms) thatis initiated by, or results from, the release of substances from primarysensory nerve terminals (such as C and A-delta fibers). As used herein,the term “secondary” neurogenic inflammation” refers to tissueinflammation initiated by non-neuronal sources (e.g., extravasation fromvascular bed or tissue interstitium-derived, such as from mast cells orimmune cells) of inflammatory mediators, such as peptides or cytokines,stimulating sensory nerve terminals and causing a release ofinflammatory mediators from the nerves. These nerve-derived inflammatorymediators can, in turn, stimulate the sensory nerves as well as actingon non-neuronal targets (e.g., mast cells). The net effect of both forms(primary and secondary) of neurogenic inflammation is to have aninflammatory state that is maintained by the sensitization of theperipheral sensory nerve fibers. The physiological consequence of theresulting neurogenic inflammation depends on the tissue in question,producing, such as, e.g., cutaneous pain (allodynia, hyperalgesia),joint arthritis, visceral pain and dysfunction, pulmonary dysfunction(asthma, COPD), and bladder dysfunction (pain, overactive bladder).

Inflammation and/or autoimmune disorder symptoms include, withoutlimitation, edema, hyperemia, erythema, bruising, tenderness, stiffness,swollenness, fever, a chill, congestion of the respiratory tractincluding nose, and bronchi, congestion of a sinus, a breathing problem,fluid retention, a blood clot, loss of appetite, increased heart rate,formation of granulomas, fibrinous, pus, or non-viscous serous fluid,formation of an ulcer, or pain. The actual symptoms associated with aninflammation and an autoimmune disorder disclosed herein are well knownand can be determined by a person of ordinary skill in the art by takinginto account factors, including, without limitation, the location of theinflammation or autoimmune disorder, the cause of the inflammation orautoimmune disorder, the severity of the inflammation or autoimmunedisorder, the tissue or organ affected by inflammation or the autoimmunedisorder, and the disorder associated with the inflammation.

Normally, inflammation serves as a protective mechanism by an organismto remove noxious stimuli as well as initiate the healing process forinjured tissue. This acute neurogenic inflammation forms the first lineof defense by maintaining tissue integrity and contributing to tissuerepair. In fact, in the absence of acute neurogenic inflammation, woundsand infections would never heal and progressive destruction of thetissue would compromise the survival of the organism. However, severe orprolonged noxious stimulation results in a chronic inflammatory responseprovoking injury rather than mediating repair. This inflammation hasbeen implicated in the pathophysiology of a wide range of unrelateddisorders which underlie a wide variety of human diseases.

Chronic inflammation and its associated symptoms can be associated witha large, unrelated group of disorders which underlie a variety of humandiseases. Non-limiting examples of disorders exhibiting inflammation asa symptom include, without limitation, acne, acid reflux/heartburn,Alzheimer's disease, appendicitis, arteritis, arthritis, asthma,allergy, allergic rhinitis, atherosclerosis, an autoimmune disorder,balanitis, blepharitis, bronchiolitis, bronchitis, bullous pemphigoid,ursitis, a cancer, carditis, celiac disease, cellulitis, cervicitis,cholangitis, cholecystitis, chorioamnionitis, chronic obstructivepulmonary disease (COPD), cirrhosis, colitis, conjunctivitis, cystitis,common cold, dacryoadenitis, dementia, dermatitis, dermatomyositis,eczema, emphysema, encephalitis, endocarditis, endometritis, enteritis,enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis,gastritis, gastroenteritis, gingivitis, glomerulonephritis, glossitis, aheart disease, hepatitis, hidradenitis suppurativa, high blood pressure,ileitis, an inflammatory dermatologic disease, an inflammatoryneuropathy, insulin resistance, interstitial cystitis, iritis, ischemicheart disease, keratitis, keratoconjunctivitis, laryngitis, mastitis,mastoiditis, a meningitis, metabolic syndrome (syndrome X), migraine,myelitis, myocarditis, myositis, nephritis, obesity, omphalitis,oophoritis, orchitis, osteochondritis, osteopenia, osteoporosis,osteitis, otitis, pancreatitis, Parkinson's disease, parotitis, pelvicinflammatory disease, pemphigus vularis, pericarditis, peritonitis,pharyngitis, phlebitis, pleuritis, pneumonitis, proctitis, prostatitis,a psoriasis, pulpitis, pyelonephritis, pylephlebitis, rheumatic fever,rhinitis, salpingitis, sialadenitis, sinusitis, a spastic colon,stomatitis, synovitis, tendonitis, tendinosis, tenosynovitis,thrombophlebitis, tonsillitis, trigonitis, a tumor, urethritis, uveitis,vaginitis, vasculitis, and vulvitis.

One type of disorder exhibiting a symptom of inflammation is anarthritis. Arthritis includes a group of conditions involving damage tothe joints of the body due to the inflammation of the synoviumincluding, without limitation osteoarthritis, rheumatoid arthritis,juvenile idiopathic arthritis, spondyloarthropathies like ankylosingspondylitis, reactive arthritis (Reiter's syndrome), psoriaticarthritis, enteropathic arthritis associated with inflammatory boweldisease, Whipple's disease and Behcet's disease, septic arthritis, gout(also known as gouty arthritis, crystal synovitis, metabolic arthritis),pseudogout (calcium pyrophosphate deposition disease), and Still'sdisease. Arthritis can affect a single joint (monoarthritis), two tofour joints (oligoarthritis) or five or more joints (polyarthritis) andcan be either an autoimmune disease or a non-autoimmune disease.

Aspects of the present disclosure provide, in part, a autoimmunedisorder. Autoimmune disorders also exhibit symptoms of inflammation.Autoimmune diseases can be broadly divided into systemic andorgan-specific autoimmune disorders, depending on the principalclinico-pathologic features of each disease. Systemic autoimmunediseases include, without limitation, systemic lupus erythematosus(SLE), Sjögren's syndrome, scleroderma, rheumatoid arthritis andpolymyositis. Local autoimmune diseases may be endocrinologic (diabetesmellitus Type 1, Hashimoto's thyroiditis, Addison's disease etc.),dermatologic (pemphigus vulgaris), hematologic (autoimmune haemolyticanemia), neural (multiple sclerosis) or can involve virtually anycircumscribed mass of body tissue. Types of autoimmune disordersinclude, without limitation, acute disseminated encephalomyelitis(ADEM), Addison's disease, an allergy, allergic rhinitis, Alzheimer'sdisease, anti-phospholipid antibody syndrome (APS), an arthritis,asthma, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmuneinner ear disease, bullous pemphigoid, cardiovascular disease, celiacdisease, Chagas disease, chronic obstructive pulmonary disease (COPD),diabetes mellitus type 1 (IDDM), eczema, endometriosis, fibromyalgia,Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS),Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathicthrombocytopenic purpura, an inflammatory bowel disease, an inflammatorydermatologic disease, interstitial cystitis, a lupus (including discoidlupus erythematosus, drug-induced lupus erythematosus, lupus nephritis,neonatal lupus, subacute cutaneous lupus erythematosus and systemiclupus erythematosus), morphea, multiple sclerosis (MS), myastheniagravis, myopathies, narcolepsy, neuromyotonia, pemphigus vulgaris,pernicious anaemia, a psoriasis, primary biliary cirrhosis, recurrentdisseminated encephalomyelitis (multiphasic disseminatedencephalomyelitis), rheumatic fever, schizophrenia, scleroderma,Sjögren's syndrome, tenosynovitis, vasculitis, and vitiligo.

Another type of disorder exhibiting a symptom of inflammation is aninflammatory myopathy. Inflammatory myopathies are caused by problemswith the immune system attacking components of the muscle, leading tosigns of inflammation in the muscle. Inflammatory myopathies include,without limitation, dermatomyositis, inclusion body myositis, andpolymyositis.

Another type of disorder exhibiting a symptom of inflammation is avasculitis. Vasculitis is a varied group of disorders featuringinflammation of a vessel wall including lymphatic vessels and bloodvessels like veins (phlebitis), arteries (arteritis) and capillaries dueto leukocyte migration and resultant damage. The inflammation may affectany size blood vessel, anywhere in the body. It may affect eitherarteries and/or veins. The inflammation may be focal, meaning that itaffects a single location within a vessel; or it may be widespread, withareas of inflammation scattered throughout a particular organ or tissue,or even affecting more than one organ system in the body. Vasculitisinclude, without limitation, Buerger's disease (thromboangiitisobliterans), cerebral vasculitis (central nervous system vasculitis),Churg-Strauss arteritis, cryoglobulinemia, essential cryoglobulinemicvasculitis, giant cell (temporal) arteritis, Golfer's vasculitis,Henoch-Schonlein purpura, hypersensitivity vasculitis (allergicvasculitis), Kawasaki disease, microscopic polyarteritis/polyangiitis,polyarteritis nodosa, polymyalgia rheumatica (PMR), rheumatoidvasculitis, Takayasu arteritis, Wegener's granulomatosis, and vasculitissecondary to connective tissue disorders like systemic lupuserythematosus (SLE), rheumatoid arthritis (RA), relapsingpolychondritis, Behcet's disease, or other connective tissue disorders,vasculitis secondary to viral infection.

Another type of disorder exhibiting a symptom of inflammation is a skindisorder. Skin disorders include, without limitation, a dermatitis,including chronic actinic dermatitis, an eczema like atopic eczema,contact eczema, xerotic eczema, seborrhoeic dermatitis, dyshidrosis,discoid eczema, venous eczema, dermatitis herpetiformis,neurodermatitis, and autoeczematization, and statis dermatitis,hidradenitis suppurativa, psoriasis including plaqure psoriasis, nailpsoriasis, guttate psoriasis, scalp psoriasis, inverse psoriasis,pustular psoriasis, and erythrodermis psoriasis, rosacea and sclerodermaincluding morphea.

Another type of disorder exhibiting a symptom of inflammation is agastrointestinal disorder. A gastrointestinal disorder includes, withoutlimitation, irritable bowel disease, an inflammatory bowel diseaseincluding Crohn's disease and an ulcerative colitis like ulcerativeproctitis, left-sided colitis, pancolitis and fulminant colitis.

Aspects of the present disclosure provide, in part, a neurodegenerativedisease. Neurodegenerative arises from the progressive loss of structureor function of neurons, including death of neurons. Non-limitingexamples of a neurodegenerative diseases include, but are not limitedto, Alexander disease, Alper's disease, Alzheimer's disease,amyloidoses, amyotrophic lateral sclerosis, anxiety, ataxiatelangiectasia, attention deficit disorders, Canavan disease, centralnervous system injuries, Charcot Marie Tooth disease, Cockayne syndrome,corticobasal degeneration, Creutzfeldt-Jakob disease, depression,encephalitis (e.g., bacterial, parasitic, fungal, or viral),Friedreich's ataxia frontotemporal dementia, hereditary spasticparaparesis, Guillain-Barre syndrome (and its variants acute motoraxonal neuropathy, acute inflammatory demyelinating polyneuropathy, andFisher syndrome), HIV/AIDS dementia complex, Huntington's disease,ischemic damage to the nervous system, Kennedy's disease, Krabbedisease, Lewy body dementia, Machado-Joseph disease, meningitis (e.g.,bacterial, parasitic, fungal, or viral), multiple sclerosis, multiplesystem atrophy, neural trauma, e.g., percussive brain damage, spinalcord injury and traumatic damage to the nervous system, a neuropathysuch as e.g., chemotherapy-induced neuropathy, diabetes-associatedneuropathy, and peripheral neuropathy, Parkinson's disease,Pelizaeus-Merzbacher disease, Pick's disease, primary lateral sclerosis,prion disorders, progressive supranuclear palsy, Refsum's disease,Sandhoff disease, schizophrenia, Schilder's disease, spinocerebellaratrophies, Steele-Richardson-Olszewski disease, stroke, tabes dorsalis,and vascular dementia. The neurodegeneration may be associated with anauto-immune disease or a non-autoimmune disease, and theneurodegeneration may be a systemic disorder or an organ-specificdisorder. Non-limiting examples of a symptom reduced by a method oftreating a neurodegenerative disease disclosed herein include anxiety,aphasia, cognition, confusion, depression, pain, paralysis, spasticity,tics, and tremors.

Aspects of the present disclosure provide, in part, reducing a symptomassociated with an autoimmune disorder or a neurodegenerative disease.In an aspect of this embodiment, the symptom reduced is inflammation,fatigue, dizziness, malaise, elevated fever and high body temperature,extreme sensitivity to cold in the hands and feet, weakness andstiffness in muscles and joints, weight changes, digestive orgastrointestinal problems, low or high blood pressure, irritability,anxiety, or depression, infertility or reduced sex drive (low libido),blood sugar changes, and depending on the type of autoimmune disease, anincrease in the size of an organ or tissue or, the destruction of anorgan or tissue.

In an embodiment, the immunosuppressive Tat derivative polypeptide hasactivity that results in increased Fas ligand (FasL) expression inantigen presenting cell regulatory macrophages (ARegs) exposed to theimmunosuppressive Tat derivative polypeptide. In aspects of thisembodiment, an immunosuppressive Tat derivative polypeptide increasesFasL expression in cells by at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, at least 200%, at least 300%, at least 400%,or at least 500%, relative to cells not exposed to the sameimmunosuppressive Tat derivative polypeptide. In other aspects of thisembodiment, an immunosuppressive Tat derivative polypeptide increasesFasL expression in cells by about 10% to about 25%, about 10% to about50%, about 10% to about 75%, about 10% to about 100%, about 10% to about200%, about 10% to about 300%, about 10% to about 400%, about 10% toabout 500%, about 25% to about 50%, about 25% to about 75%, about 25% toabout 100%, about 25% to about 200%, about 25% to about 300%, about 25%to about 400%, about 25% to about 500%, about 50% to about 100%, about50% to about 200%, about 50% to about 300%, about 50% to about 400%, orabout 50% to about 500%, relative to cells not exposed to the sameimmunosupppressive Tat derivative. In certain embodiments, the ARegs areCD14+ macrophages.

The disclosed autoimmune, neurodegenerative, and inflammation-associateddiseases are immune-mediated disorders characterized by an increase in Tcell-mediated inflammation that is desired to be reduced. A therapeuticagent which increases, or induces expression of, FasL on ARegs, ordendritic cells, decreases T cell-mediated inflammation and therebytreats the autoimmune, neurodegenerative, or inflammation associateddisease. Therefore, in one embodiment, the present disclosure provides amethod for decreasing T cell-mediated inflammation in a subject with anautoimmune, neurodegenerative, or inflammation-associated disease byadministration of one or more of the disclosed immunosuppressive Tatderivative polypeptides, thereby treating the autoimmune,neurodegenerative, or inflammation-associated disease. In anotherembodiment, the present disclosure provides a method of inducing theexpression of FasL on ARegs in a subject with an autoimmune,neurodegenerative, or inflammation-associated disease by administrationof one or more of the disclosed immunosuppressive Tat derivativepolypeptides, thereby treating the autoimmune, neurodegenerative, orinflammation-associated disease.

Aspects of the present disclosure additionally provide, in part,reducing a symptom associated with inflammation. In an aspect of thisembodiment, the symptom reduced is edema, hyperemia, erythema, bruising,tenderness, stiffness, swollenness, fever, a chill, congestion of therespiratory tract including nose, and bronchi, congestion of a sinus, abreathing problem, fluid retention, a blood clot, a loss of appetite, anincreased heart rate, a formation of granulomas, fibrinous, pus, ornon-viscous serous fluid, a formation of an ulcer, or pain.

Aspects of the present disclosure provide, in part, a mammal. A mammalincludes a human, and a human can be a patient. Other aspects of thepresent disclosure provide, in part, an individual. An individualincludes a mammal and a human, and a human can be a patient. In otherembodiments, mammals include domesticated animals such as dogs, cats,horses, cattle, sheep, goats, and primates.

Aspects of the present disclosure provide, in part, administering acompound or a composition disclosed herein. As used herein, the term“administering” means any delivery mechanism that provides a compound ora composition disclosed herein to an individual that potentially resultsin a clinically, therapeutically, or experimentally beneficial result.

Administration of a compound or a composition disclosed herein include avariety of enteral or parenteral approaches including, withoutlimitation, oral administration in any acceptable form, such as, e.g.,tablet, liquid, capsule, powder, or the like; topical administration inany acceptable form, such as, e.g., drops, spray, creams, gels orointments; buccal, nasal, and/or inhalation administration in anyacceptable form; rectal administration in any acceptable form; vaginaladministration in any acceptable form; intravascular administration inany acceptable form, such as, e.g., intravenous bolus injection,intravenous infusion, intra-arterial bolus injection, intra-arterialinfusion and catheter instillation into the vasculature; peri- andintra-tissue administration in any acceptable form, such as, e.g.,intraperitoneal injection, intramuscular injection, subcutaneousinjection, subcutaneous infusion, intraocular injection, retinalinjection, or sub-retinal injection or epidural injection;intravesicular administration in any acceptable form, such as, e.g.,catheter instillation; and by placement device, such as, e.g., animplant, a stent, a patch, a pellet, a catheter, an osmotic pump, asuppository, a bioerodible delivery system, a non-bioerodible deliverysystem or another implanted extended or slow release system. Anexemplary list of biodegradable polymers and methods of use aredescribed in, e.g., Handbook of Biodegradable Polymers (Abraham J. Dombet al., eds., Overseas Publishers Association, 1997).

A compound or a composition disclosed herein can be administered to amammal using a variety of routes. Routes of administration suitable fortreating inflammation, an autoimmune disorder, or a neurodegenerativedisease as disclosed herein include both local and systemicadministration. Local administration results in significantly moredelivery of a composition to a specific location as compared to theentire body of the mammal, whereas, systemic administration results indelivery of a composition to essentially the entire body of theindividual. Suitable routes of administration also include both centraland peripheral administration. Central administration results indelivery of a compound or a composition to essentially the centralnervous system of the individual and includes, e.g., intrathecaladministration, epidural administration as well as a cranial injectionor implant. Peripheral administration results in delivery of a compoundor a composition to essentially any area of an individual outside of thecentral nervous system and encompasses any route of administration otherthan direct administration to the spine or brain. The actual route ofadministration of a compound or a composition disclosed herein used canbe determined by a person of ordinary skill in the art by taking intoaccount factors, including, without limitation, the type, location,cause and severity of inflammation or an autoimmune or neurodegenerativedisorder, the duration of treatment desired, the degree of reliefdesired, the duration of relief desired, the particular compound orcomposition used, the rate of excretion of the compound or compositionused, the pharmacodynamics of the compound or composition used, thenature of the other compounds to be included in the composition, theparticular route of administration, the particular characteristics,history and risk factors of the individual, such as, e.g., age, weight,general health and the like, the response of the individual to thetreatment, or any combination thereof. An effective dosage amount of acompound or a composition disclosed herein can thus readily bedetermined by the person of ordinary skill in the art considering allcriteria and utilizing his best judgment on the individual's behalf.

In an embodiment, a compound or a composition disclosed herein isadministered systemically to a mammal. In another embodiment, a compoundor a composition disclosed herein is administered locally to a mammal.In an aspect of this embodiment, a compound or a composition disclosedherein is administered to a site of inflammation, neurodegeneration, orautoimmune disorder of a mammal. In another aspect of this embodiment, acompound or a composition disclosed herein is administered to the areasurrounding an inflammation, neurodegenerative disease, or autoimmunedisorder of a mammal.

Aspects of the present specification provide, in part, administering atherapeutically effective amount of a compound or a compositiondisclosed herein. As used herein, the term “therapeutically effectiveamount” is synonymous with “therapeutically effective dose” and whenused in reference to treating inflammation, neurodegeneration, or anautoimmune disorder means the minimum dose of a compound or compositiondisclosed herein necessary to achieve the desired therapeutic effect andincludes a dose sufficient to reduce a symptom associated withinflammation, neurodegeneration, or an autoimmune disorder. In aspectsof this embodiment, a therapeutically effective amount of a compound ora composition disclosed herein reduces a symptom associated withinflammation, neurodegeneration, or an autoimmune disorder by, e.g., atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or at least 100%. Inother aspects of this embodiment, a therapeutically effective amount ofa compound or a composition disclosed herein reduces a symptomassociated with inflammation, neurodegeneration, or an autoimmunedisorder by, e.g., at most 10%, at most 20%, at most 30%, at most 40%,at most 50%, at most 60%, at most 70%, at most 80%, at most 90% or atmost 100%. In yet other aspects of this embodiment, a therapeuticallyeffective amount of a compound or a composition disclosed herein reducesa symptom associated with inflammation, neurodegeneration, or anautoimmune disorder by, e.g., about 10% to about 100%, about 10% toabout 90%, about 10% to about 80%, about 10% to about 70%, about 10% toabout 60%, about 10% to about 50%, about 10% to about 40%, about 20% toabout 100%, about 20% to about 90%, about 20% to about 80%, about 20% toabout 20%, about 20% to about 60%, about 20% to about 50%, about 20% toabout 40%, about 30% to about 100%, about 30% to about 90%, about 30% toabout 80%, about 30% to about 70%, about 30% to about 60%, or about 30%to about 50%. In still other aspects of this embodiment, atherapeutically effective amount of a compound or a compositiondisclosed herein is the dosage sufficient to reduces a symptomassociated with inflammation, neurodegeneration, or an autoimmunedisorder for, e.g., at least one week, at least one month, at least twomonths, at least three months, at least four months, at least fivemonths, at least six months, at least seven months, at least eightmonths, at least nine months, at least ten months, at least elevenmonths, or at least twelve months.

The amount of active component in a compound or a composition disclosedherein for treating inflammation, neurodegeneration, or an autoimmunedisorder can be varied so that a suitable dosage is obtained. The actualtherapeutically effective amount of a compound or a compositiondisclosed herein to be administered to a mammal can be determined by aperson of ordinary skill in the art by taking into account factors,including, without limitation, the type, location, cause or severity ofinflammation, neurodegeneration, or an autoimmune disorder, the durationof treatment desired, the degree of relief desired, the duration ofrelief desired, the particular compound or composition used, the rate ofexcretion of the compound or composition used, the pharmacodynamics ofthe compound or composition used, the nature of the other compounds tobe included in the composition, the particular route of administration,the particular characteristics, history and risk factors of theindividual, such as, e.g., age, weight, general health and the like, theresponse of the individual to the treatment, or any combination thereof.An effective dosage amount of a compound or a composition disclosedherein can thus readily be determined by the person of ordinary skill inthe art considering all criteria and utilizing his best judgment on theindividual's behalf.

Additionally, where repeated administration of a compound or acomposition disclosed herein is used, the actual effect amount of acompound or a composition disclosed herein will further depend uponfactors, including, without limitation, the frequency of administration,the half-life of the compound or composition disclosed herein, or anycombination thereof. It is known by a person of ordinary skill in theart that an effective amount of a compound or a composition disclosedherein can be extrapolated from in vitro assays and in vivoadministration studies using animal models prior to administration tohumans. Wde variations in the necessary effective amount are to beexpected in view of the differing efficiencies of the various routes ofadministration. For instance, oral administration generally would beexpected to require higher dosage levels than administration byintravenous or intravitreal injection. Variations in these dosage levelscan be adjusted using standard empirical routines of optimization, whichare well-known to a person of ordinary skill in the art. The precisetherapeutically effective dosage levels and patterns are preferablydetermined by the attending physician in consideration of theabove-identified factors.

Dosing can be single dosage or cumulative (serial dosing), and can bereadily determined by one skilled in the art. For instance, treatment ofinflammation, neurodegeneration, or an autoimmune disorder may comprisea one-time administration of an effective dose of a compound or acomposition disclosed herein. As a non-limiting example, an effectivedose of a compound or a composition disclosed herein can be administeredonce to a mammal, e.g., as a single injection or deposition at or nearthe site exhibiting a symptom of inflammation, neurodegeneration, or anautoimmune disorder or a single oral administration of the compound or acomposition. Alternatively, treatment of inflammation,neurodegeneration, or an autoimmune disorder may comprise multipleadministrations of an effective dose of a compound or a compositiondisclosed herein carried out over a range of time periods, such as,e.g., daily, once every few days, weekly, monthly or yearly. As anon-limiting example, a compound or a composition disclosed herein canbe administered once or twice weekly to a mammal. The timing ofadministration can vary from mammal to mammal, depending upon suchfactors as the severity of a mammal's symptoms. For example, aneffective dose of a compound or a composition disclosed herein can beadministered to a mammal once a month for an indefinite period of time,or until the mammal no longer requires therapy. A person of ordinaryskill in the art will recognize that the condition of the mammal can bemonitored throughout the course of treatment and that the effectiveamount of a compound or a composition disclosed herein that isadministered can be adjusted accordingly.

A compound or a composition disclosed herein as disclosed herein canalso be administered to a mammal in combination with other therapeuticcompounds to increase the overall therapeutic effect of the treatment.The use of multiple compounds to treat autoimmune, neurodegenerative,and inflammation-associated diseases is within the scope of the presentdisclosure. Additionally, the present disclosure includes the use of thedisclosed peptides to treat autoimmune, neurodegenerative, andinflammation-associated diseases and the use of the disclosed peptidesin the manufacture of a medicament to treat autoimmune,neurodegenerative, and inflammation-associated diseases.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification, including those pertaining to the methods of treating anautoimmune disorder, inflammation, or neurodegenerative diseases usingthe Tat derivatives disclosed herein.

Example 1 Effects of Tat on the Dendritic Cell Lineage

Tat induces monocytes committed to the dendritic cell (DC) lineage toenlarge into activated, CD86+ DC APCs (FIG. 1). Human monocytes enrichedfrom PBMCs by Percoll density gradient separation and adherance toanti-CD14 coated magnetic beads (Dynabeads M-450, Dynal Biotech) werecommitted to differentiate into DCs through five days of culture inGM-CSF (100 ng/mL) and IL-4 (100 ng/mL). Committed DCs were culturedovernight either in medium alone (Control), LPS (100 ng/mL), or Tat (50nM), after which they were stained with an anti-CD86 antibody (BDPharmingen) and analyzed by FACScan for CD86 induction (left panel) orgeneralized activation (right panel, enlargement into box R2, shown forTat-stimulated cells). The MFIs for CD86 expression are 9 (Control), 30(LPS), and 187 (Tat), CD86 being a specific determinant of DCactivation.

Derivitzed Tat reduces AReg differentiation and potently enhancesantigen-specific activation of CTLs (FIG. 2). Tat is chemicallyderivatized by oxidation (Tat* or ox-Tat) so that it does not induceARegs from monocyte APC precursors (FIG. 3). Ten micrograms of Tat/p24Tat*-Ag conjugate (Ag-Tat*) was administered into the flanks of Balb/Cmice in adjuvant on day 0 and day 7. Experimental groups werecomparatively immunized in adjuvant with 5 μg of p24 in one flank and 5μg derivatized Tat in the other flank (Ag & Tat*), or 10 μg of p24 inadjuvant (Ag). Control mice were given two injections of adjuvant. Fourmice were treated in each group. At day 14, draining lymph node cellsfrom each animal were harvested and re-stimulated overnight in culturesof irradiated Ap24 (H-2d cells stably transfected to express antigenp24) cells or control non-transfected cells. CTL activity wasquantitated as the number of γ-interferon secreting spot formingcolonies (SFC)/10⁶ plated cells using ELISPOT assays. The backgroundwith non-transfected re-stimulators, which was in all cases<10 SFC/10⁶,is subtracted from each point. The results are indicative of threesimilar experiments.

Example 2 Tat Activation of Macrophages and Suppression of the ImmuneResponse

Recombinant Tat protein is prepared as previously described (Li, C. J.et al. (1995), Science 268:429-31) under mildly denaturing conditionsand was renatured in the presence of 0.1 mM DTT.

Tat activation of monocytes is dose-dependent and saturatable (FIG. 3).Human monocytes were cultured in increasing concentrations ofrecombinant Tat for six days at which time they were assayed for Fasligand (FasL) induction as a measure of activation by using flowcytometry (FACScan, Becton Dickinson) to quantitate the intensity ofstaining (mean fluorescence index (MFI)) with an anti-Fas ligandmonoclonal antibody (Nok 1, BD Pharmingen). Higher concentrations of Tatdid not increase MFI (not shown), and T cells could not be activatedwith 50 nM Tat (not shown), the plateau stimulatory concentration forAPCs.

Tat suppresses the antigen-specific humoral immune response to HIV-1 p24(FIG. 4). At week 0, mice (4 in each group) were immunized with 5 μgrecombinant p24 protein (Chiron, Emeryville, Calif.) and either 5 μgrecombinant Tat protein (PT) or 5 μg recombinant ox-Tat* protein (Ag)mixed in 100 μL complete Freund's adjuvant and administeredsubcutaneously in the flank. Following immunization, sera were collectedevery other week for 10 weeks and assayed for a specific antibodyresponse to p24 by commercially available ELISA (Abbott Laboratories,Abbott Park, Ill.). The p24 antibody titer at 2 weeks (FIG. 4A) wascompletely suppressed by the Tat protein (PT) compared with the ox-Tat*control (Ag). This response was maintained for at least 6 weeks. Theantibody titers at 6 weeks are approximately ten times greater than atweek 2 due to maturation of the immune response.

Tat enhances the viability of cultured murine macrophages as long as themacrophages were first activated in vivo compared with no prioractivation and stimulated with relatively high concentrations of Tat(FIG. 5). APCs were isolated by peritoneal lavage from miceintraperitoneally injected four days earlier with either 2.9%thioglycolate (as adjuvant) or 0.85% saline solution (resting).Harvested washout cells were cultured at 10⁶ cells/mL for five days inmedium alone (Control, C), lipopolysaccharide (LPS, 100 ng/mL), or Tatproduced as recombinant protein in E. coli (Tat, 500 ng/mL). Activationwas determined as % enlarged cells (M1 fraction).

The immunosuppressive Tat produces a stable suppression of mouselymphocyte proliferation (FIG. 6). Mice were immunized in quadruplicatewith a Freund's adjuvant emulsion containing either 5 μg Tat/p24(recombinant HIV-1 gag protein p24) tolerogen (GRP 2) or with 5 μgavidin-p24 (GRP 1) as control. At two weeks residual draining lymph nodecells were harvested, pooled within each group, and cultured at 10⁵cells/microtiter well for four days in the presence of gradedconcentrations of recombinant p24 protein (p24, μg/mL). Proliferationwas assayed as a determinant of recall T cell response by quantitatingovernight ³H thymidine uptake (CPM) in a liquid scintillation counter.This response is maintained for up to six weeks.

In addition, the immunosuppressive Tat generates an antigen-specificimmune suppression (FIG. 7). Mice in quadruplicate were immunized at day0 and boosted at day 7 with an adjuvant emulsion containing either 5 μgTat/p24 tolerogen (Ag+Tol) or with 5 μg avidin-p24 (Ag Alone) ascontrol. At day 14, draining lymph node cells were harvested andstimulated at 10⁵ cells/microtiter culture well either with addedantigen (Specific, recombinant p24, 1 μg/mL) or with added anti-T cellreceptor monoclonal antibody (NonSpecific, 2C11, 10 μg/mL). Tritiatedthymidine uptake (CPM) was determined by liquid scintillation at day 4of culture. The specific Ag+Tol response is suppressed 98% relative toAg alone, and is not distinguishable from cells cultured in the absenceof stimulants.

Example 3 Tat Suppression is Mediated by ARegs

Tat mediated antigen-specific suppression is mediated throughtrans-(intracellular) activation of a CD14+ FasL+ macrophage (FIG. 8).In mice, Tat tolerizes at the T cell level and is maintained for atleast six weeks after the initial treatment under the conditionsdemonstrated in FIG. 6. A human peripheral blood mononuclear cell (PBMC)population enriched for monocytes by Percoll centrifugation was culturedfor four days either in medium containing 5% fetal calf serum (FCS,Control), Tat (50 nM), or LPS (100 ng/mL). Harvested cells were doublystained with a fluoresceinated (anti-fl1) anti-FasL monoclonal antibody(Mab), (αFasL-FITC, Nok 1, BD Pharmingen) and with an anti-CD14rhodamine labeled Mab (αCD14fl2, BD Biosciences, CD14 being adeterminant specific to macrophages (Mϕ). Cells were analyzed by FACScan(Becton Dickinson) for activation (Forward Scatter), CD14 expression(R2, percent Mϕs), and for induction of FasL (MFI). The T cellpopulation (R1) was CD14− and did not express FasL. Similar results wereobtained from cells harvested after 2, 3, 5, or 6 days of culture as forPBMCs harvested at day four.

In human cells, Tat-activated macrophages are regulatory andimmunosuppressive APC macrophage regulators (ARegs) (FIG. 9). To definethe pathway of Tat immunosuppression, through FasL induction on themacrophage, resulting in loss of helper T cell recall responses, T cellproliferation assays were used with recall antigens, tat and FasLantagonists. In FIG. 9A, human PBMCs from one individual were culturedin triplicate for 5 days in either medium (not shown), tetanus antigen(Ag, 0.3 Lf/mL), antigen with the further addition of 50 nM Tat (Ag+Tat)or Ag with 50 nM Tat and recombinant sFas protein (25 μg/mL) to blocksurface Fas L expressed on macrophages (Ag+Tat+sFas). Tritiatedthymidine was added over the last 18 hours, and results are graphed asstimulation index (mean cpm stimulated culture/mean cpm medium control).Results are representative of three similar experiments. At lowconcentrations of Tat (50 nM), Tat-induced immunosuppression was notonly fully reversed by the addition of soluble Fas, but under theseconditions, Tat actually became stimulatory (141% relative to antigentreatment alone). FIG. 9B: Proliferation of PBMCs cultured 6 days witheither tetanus or Candida antigen alone (Ag), compared with cultures inwhich Tat (Ag+Tat, 125 nM), or Tat (125 nM) and the antagonisticanti-Fas antibody, ZB4 (250 μg/mL, Upstate Biotechnology) also wereadded (Ag+Tat+αFas). Results are representative of three similarexperiments.

Example 4 In Vitro Bioassay for Monocyte Differentiation

The in vitro ultra-sensitive monocyte Tat bioassay is used to assess theimmunosuppressant or immunostimulatory activity of the Tat proteinsdisclosed herein. This assay utilizes fresh monocyte cells substantiallypurified from human peripheral blood using standard density gradientenrichment procedures or other cell isolation protocols known in theart. The substantially purified monocytes are washed and then culturedin RPMI-1640 supplemented with 10% FBS at 37° C.

The in vitro ultra-sensitive monocyte Tat bioassay is performed using apositive control (FasL, inducing compound) and a negative control (noactive compound is added to the culture). Additional suitable positivecontrols include, but are not limited to, lipopolysaccharide (LPS) andor tumor necrosis factor (TNF-α) at a final concentration of 100 ng/mLand 50 ng/mL, respectively. Test samples (Tat preparations) are run atfinal concentrations from 50 pM to 50 nM and include Tat, ox-Tat, andother Tat derivatives and mutants.

The test samples and controls are individually mixed with thesubstantially pure monocytes seeded at a density of 10⁶ cells/mL inround bottom tubes containing RPMI-1640 with 10% FBS (herein referred tocollectively as assay cultures). The assay cultures are then incubatedfor a suitable period of time, preferably from five to six days, at 37°C., in a 5% CO₂ environment.

At the end of the incubation period, cells are removed from each assayculture and the presence of any induced FasL expression (for measurementof differentiation into ARegs) or CD86 expression (for differentiationin dendritic cells) is detected by staining with anti-FasL or anti-CD86antibodies and appropriate fluorescent detection agents. After thecultures have been stained, the fluorescence is detected using afluorescence activated cell sorter (FACS) system. Control staining isperformed using the fluorescent detection system alone and subtractedfrom the specific anti-FasL or anti-CD86 staining seen in the assaycultures. The greater the percentage of FasL positive cells in a givenassay culture, the more immunosuppressant the test sample in the assayculture is. Conversely, if the assay culture contains a predominance ofCD86 positive cells, the test sample is identified to beimmunostimulatory. Negative controls should always remain non-reactivewith the antibodies and the positive control should fall withinpredetermined ranges.

Example 5 Synthesis of Tat Derivatives

Synthetic peptides were assembled by standard Fmoc chemistry using aCS336X automated synthesizer (C S Bio Co., Menlo Park, Calif.). Thepeptides were cleaved from the resin using trifluoroacetic acidcleavage/deprotection cocktail compatible with the presence of sevencysteines. Purification was done using reverse phase HPLC. The finalproduct was lyophilized from H₂O/acetonitrile. A portion of eachsynthesis was labeled with Alexa-488. All synthetic Tat derivativesachieved a purity of >95%. Synthetic peptides were reconstituted inphosphate buffer in the presence of 0.1 mM dithiothreitol before use.

Example 6 In Vitro Activity of Tat Derivatives

Human monocytes were cultured for 24-28 hours with a Tat derivative (SEQID NO:7, Nani-P2), (FIG. 10) or lipopolysaccharide (LPS) (FIG. 11) andthe cells were then washed and stained with fluorescent-labeled CD86.The Tat derivative stimulated higher expression of CD86 than either ISS(TLR) or LPS.

Example 7 Live Cell Imaging of Tat Derivatives in T-Lymphocyte andMonocyte Cell Lines

A Jurkat cell line (Clone E6-1, American Type Culture Collection) wasmaintained in RPMI-1640 (GIBCO), 10% heat-inactivated FBS (GIBCO), 50 Upenicillin/50 μg/ml streptomycin (GIBCO), and 2 mM L-Glutamine (GIBCO)at 37° C. in 5% CO₂. U937 Cells (American Type Culture Collection) weremaintained in RPMI-1640 (GIBCO), 10% heat-inactivated FBS, 50 Upenicillin, 50 μg/ml streptomycin, and 2 mM L-Glutamine (GIBCO), 10 mMHEPES (GIBCO), and 0.005% β-mercaptoethanol (Sigma-Aldrich) at 37° C. in5% CO₂.

One million live Jurkat or U937 cells were incubated in complete culturemedium for 60-120 minutes at 37° C. in 5% CO₂, with or without additionof 5 μM Alexa-488-labeled synthetic Tat derivative. Cells were washed inPBS containing 0.1% heat-inactivated FBS, 1 mM EDTA, and stained with aHOECHST-NucBlue Live Cell Stain (Molecular Probes) by adding 2 drops/mLto the cell suspension. Cells were kept on ice until live cell imagingwas performed.

Cells were placed in glass-bottom dishes (MatTek Corp.) and imaged on aZeiss Axio Observer.Z1 microscope (Carl Zeiss) using a Plan-Neofluor100×/1.3 objective lens. LEDs (Colibri; Carl Zeiss) at 365 nm and 470 nmwere used to excite HOESCHT-stained DNA and Alexa488-labeled protein.Emission was collected with standard DAPI and FITC fluorescence filters.Fluorescence and phase-contrast images were collected with an Orca ERcooled CCD camera (Hamamatsu Photonics).

The results, depicted in FIGS. 12 and 13 demonstrate the ability ofsynthetic Tat derivatives to enter T-lymphocyte and monocytic cell linesin vitro. Jurkat T-cells (FIG. 12) and U937 monocytes (FIG. 13) treatedwith Alexa488-labeled Tat derivatives show cellular uptake and nuclearlocalization of fluorescently labeled proteins, highlighting theapplication of these molecules as cell penetrating peptides endowed withthe ability to localize within and around the nucleus.

Example 8 Fluorescence Activated Cell Sorting of Tat Derivative TreatedCells

Human peripheral blood mononuclear cells (PBMC) were isolated from wholeblood by density gradient centrifugation with Histopaque separationmedium using an ACCUSPIN™ centrifuge tube (Sigma-Aldrich). PBMC cultureswere established in RPMI-1640, 10% heat-inactivated fetal bovine serum,50 U penicillin/50 μg/mL streptomycin, and 2 mM L-Glutamine at a celldensity of 1×10⁶ cells/mL in a 12 well tissue culture plate. PBMC's wereincubated in the presence of either no stimulus, 1 μg/ml LPS(Sigma-Aldrich), 100 ng/ml TNF-α (Peprotech), or 1 μg/ml synthetic Tatderivative (SEQ ID NO:9) at 37° C. in 5% CO₂. After 3-5 days, cells wereharvested, washed, and stained with a mouse anti-humanCD14-allophycocyanin (APC) conjugated monoclonal antibody (clone M5E2,BD Biosciences,) or a mouse anti-human Fas Ligand fluorescein (FITC)conjugated monoclonal antibody (clone SB93a, Southern Biotech).Propidium iodide was added to assess viability before fluorescenceactivated cell sorting (FACS) was performed on a BD LSR II FlowCytometer (BD Biosciences).

The results, depicted in FIG. 14-17 demonstrate the ability of Tatderivatives to stimulate and increase cell surface CD14 and FasL onhuman PBMC's in vitro relative to non-stimulated controls by FACSindicating the presence AReg cells capable of suppressing a T-cellimmune response.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

What is claimed is:
 1. A method of treating a disease characterized byaberrant immune responses, the method comprising: administering atherapeutically effective amount of one or more immunosuppressivepolypeptides to a subject in need thereof, wherein the immunosuppressivepolypeptide comprises: (A) an amino acid sequence comprising thefollowing domains in the indicated order: (i) a transcription factor(TF) domain comprising a sequence from an immunosuppressive humanimmunodeficiency virus (HIV) or simian immunodeficiency virus (SIV)transactivator of transcription (Tat) protein, hairless or an artificialimmunosuppressive sequence, wherein the TF domain comprises the aminoacid sequence of one of SEQ ID NOs: 36, 39, 44, 48, 50, or 54; (ii) acysteine-rich region from lentiviral Tat or a defensin molecule, whereinthe cysteine-rich region comprises the amino acid sequence of one of SEQID NOs: 37, 40, 41, 43, 45, 51, 55, 57, 58, 62, 63, 64, or 70; and (iii)a C-terminal region from a lentiviral Tat protein comprising the aminoacid sequence of one of SEQ ID NOs: 38, 42, 46, 52, 68, or 71; (B) atranscription factor (TF) domain according to (i), and a cysteine-richdomain and C-terminal domain, in that order, wherein the cysteine-richdomain and C-terminal domain together comprise an amino acid sequence ofone of SEQ ID NOs: 47, 49 or 53; or (C) or a conservative variant of theimmunosuppressive polypeptide of (A) or (B) having at least 95% sequenceidentity thereto; and thereby treating the disease by suppressing theimmune system.
 2. The method of claim 1, further comprising anarginine-rich domain from a lentiviral Tat protein.
 3. The method ofclaim 1, wherein the lentiviral Tat is from HIV-1, HIV-2, SIV, felineimmunodeficiency virus (FIV), or equine infectious anemia virus (EIAV).4. The method of claim 1, wherein the TF domain further comprises arepeat sequence comprising (PVDPRLEPWKHPGSQP)_(n) (SEQ ID NO: 12) at theN-terminus, wherein n=2-10.
 5. The method of claim 1, wherein theimmunosuppressive Tat derivative polypeptide comprises the amino acidsequence of any of SEQ ID NOs: 9-11, 13-35, or
 69. 6. The method ofclaim 1, wherein the treatment increases the expression of Fas ligand onantigen presenting cell regulatory macrophages (ARegs).
 7. The method ofclaim 1, wherein the disease is an autoimmune, neurodegenerative orinflammation-associated disorder.
 8. The method of claim 7, wherein theautoimmune disorder is an acute disseminated encephalomyelitis (ADEM),an Addison's disease, an allergy, allergic rhinitis, an Alzheimer'sdisease, an anti-phospholipid antibody syndrome (APS), an arthritis, anasthma, an autoimmune deficiency syndrome, an autoimmune hemolyticanemia, an autoimmune hepatitis, an autoimmune inner ear disease, abullous pemphigoid, a celiac disease, a Chagas disease, a chronicobstructive pulmonary disease (COPD), a diabetes mellitus type 1 (IDDM),an eczema, an endometriosis, a gastrointestinal disorder, aGoodpasture's syndrome, a Graves' disease, a Guillain-Barré syndrome(GBS), a Hashimoto's thyroiditis, a hidradenitis suppurativa, anidiopathic thrombocytopenic purpura, an inflammatory bowel disease, aninflammatory dermatologic disease, an interstitial cystitis, a lupus, amorphea, a multiple sclerosis (MS), a myasthenia gravis, a myopathy, anarcolepsy, a neuromyotonia, a pemphigus vulgaris, a pernicious anaemia,a primary biliary cirrhosis, a psoriasis, a recurrent disseminatedencephalomyelitis, a rheumatic fever, a schizophrenia, a scleroderma, aSjögren's syndrome, a skin disorder, a tenosynovitis, a uveitis, avasculitis, or a vitiligo.
 9. The method of claim 7, wherein the diseaseassociated with inflammation is an acne, an acid reflux/heartburn, anallergy, an allergic rhinitis, an Alzheimer's disease, an appendicitis,an arteritis, an arthritis, an asthma, an atherosclerosis, an autoimmunedisorder, a balanitis, a blepharitis, a bronchiolitis, a bronchitis, abursitis, a cancer, a carditis, a celiac disease, a cellulitis, acervicitis, a cholangitis, a cholecystitis, a chorioamnionitis, achronic obstructive pulmonary disease (COPD), a cirrhosis, a colitis, aconjunctivitis, a cystitis, a common cold, a dacryoadenitis, a dementia,a dermatitis, a dermatomyositis, an eczema, an emphysema, anencephalitis, an endocarditis, an endometritis, an enteritis, anenterocolitis, an epicondylitis, an epididymitis, a fasciitis, afibrositis, a gastritis, a gastroenteritis, a gingivitis, aglomerulonephritis, a glossitis, a heart disease, a hepatitis, ahidradenitis suppurativa, a high blood pressure, an ileitis, an insulinresistance, an interstitial cystitis, an iritis, an ischemic heartdisease, a keratitis, a keratoconjunctivitis, a laryngitis, a lupus, amastitis, a mastoiditis, a meningitis, a metabolic syndrome (syndromeX), a migraine, a multiple sclerosis, a myelitis, a myocarditis, amyopathy, a myositis, a nephritis, a neuropathy, an obesity, anomphalitis, an oophoritis, an orchitis, an osteochondritis, anosteopenia, an osteoporosis, an osteitis, an otitis, a pancreatitis, aParkinson's disease, a parotitis, a pelvic inflammatory disease, apericarditis, a peritonitis, a pharyngitis, a phlebitis, a pleuritis, apneumonitis, a proctitis, a prostatitis, a psoriasis, a pulpitis, apyelonephritis, a pylephlebitis, a rheumatic fever, a rhinitis, asalpingitis, a sialadenitis, a sinusitis, a spastic colon, a stomatitis,a synovitis, a tendonitis, a tendinosis, a tenosynovitis, athrombophlebitis, a tonsillitis, a trigonitis, a tumor, an urethritis,an uveitis, a vaginitis, a vasculitis, or a vulvitis.
 10. The method ofclaim 7, wherein the neurodegenerative disease is Alexander disease,Alper's disease, Alzheimer's disease, amyloidoses, amyotrophic lateralsclerosis, anxiety, ataxia telangiectasia, attention deficit disorders,Canavan disease, central nervous system injuries, Charcot Marie Toothdisease, Cockayne syndrome, corticobasal degeneration, Creutzfeldt-Jakobdisease, depression, encephalitis (e.g., bacterial, parasitic, fungal,or viral), Friedreich's ataxia frontotemporal dementia, hereditaryspastic paraparesis, Guillain-Barre syndrome (and its variants acutemotor axonal neuropathy, acute inflammatory demyelinatingpolyneuropathy, and Fisher syndrome), HIV/AIDS dementia complex,Huntington's disease, ischemic damage to the nervous system, Kennedy'sdisease, Krabbe disease, Lewy body dementia, Machado-Joseph disease,meningitis (e.g., bacterial, parasitic, fungal, or viral) multiplesclerosis, multiple system atrophy, neural trauma, e.g., percussivebrain damage, spinal cord injury and traumatic damage to the nervoussystem, a neuropathy such as e.g., chemotherapy-induced neuropathy,diabetes-associated neuropathy, and peripheral neuropathy, Parkinson'sdisease, Pelizaeus-Merzbacher disease, Pick's disease, primary lateralsclerosis, prion disorders, progressive supranuclear palsy, Refsum'sdisease, Sandhoff disease, schizophrenia, Schilder's disease,spinocerebellar atrophies, Steele-Richardson-Olszewski disease, stroke,tabes dorsalis, or vascular dementia.
 11. The method of claim 1, whereinthe method further causes the reduction of at least one symptomassociated with the autoimmune disease, neurodegenerative disease, ordisease associated with inflammation and therein the symptom isinflammation, fatigue, dizziness, malaise, elevated fever and high bodytemperature, extreme sensitivity to cold in the hands and feet, weaknessand stiffness in muscles and joints, weight changes, digestive orgastrointestinal problems, low or high blood pressure, irritability,anxiety, or depression, infertility or reduced sex drive (low libido),blood sugar changes, and depending on the type of autoimmune disease, anincrease in the size of an organ or tissue, or the destruction of anorgan or tissue.
 12. The method of claim 1, wherein, as a result of theadministration, secretion of cytokines by ARegs is decreased.
 13. Themethod of claim 1, wherein the immunosuppressive Tat derivativepolypeptide is administered in a plurality of doses.
 14. The method ofclaim 13, wherein the immunosuppressive Tat derivative polypeptide isadministered daily, weekly, biweekly, monthly, or bimonthly.
 15. Themethod of claim 1, wherein the administering step comprises a repetitiveadministration cycle wherein each cycle comprises administering aplurality of doses of the immunosuppressive Tat derivative polypeptidein a defined time period followed by a rest period and wherein the cycleis repeated a plurality of times.
 16. A method of increasing theexpression of Fas ligand (FasL) on antigen presenting cell regulatorymacrophages (ARegs), the method comprising: administering atherapeutically effective amount of one or more immunosuppressivepolypeptides to a subject in need thereof, wherein the immunosuppressivepolypeptide comprises: (A) an amino acid sequence comprising thefollowing domains in the indicated order: (i) a transcription factor(TF) domain comprising a sequence from an immunosuppressive humanimmunodeficiency virus (HIV) or simian immunodeficiency virus (SIV)transactivator of transcription (Tat) protein, hairless or an artificialimmunosuppressive sequence, wherein the TF domain comprises the aminoacid sequence of one of SEQ ID NOs: 36, 39, 44, 48, 50, or 54; (ii) acysteine-rich region from lentiviral Tat or a defensin molecule, whereinthe cysteine-rich region comprises the amino acid sequence of one of SEQID NOs: 37, 40, 41, 43, 45, 51, 55, 57, 58, 62, 63, 64, or 70; and (iii)a C-terminal region from a lentiviral Tat protein comprising the aminoacid sequence of one of SEQ ID NOs: 38, 42, 46, 52, 68, or 71; (B) atranscription factor (TF) domain according to (i), and a cysteine-richdomain and C-terminal domain, in that order, wherein the cysteine-richdomain and C-terminal domain together comprise an amino acid sequence ofone of SEQ ID NOs: 47, 49 or 53; or (C) or a conservative variant of theimmunosuppressive polypeptide of (A) or (B) having at least 95% sequenceidentity thereto; and thereby increasing the expression of FasL on theARegs.
 17. A method of reducing inflammation, the method comprising:administering a therapeutically effective amount of one or moreimmunosuppressive polypeptides to a subject in need thereof, wherein theimmunosuppressive polypeptide comprises: (A) an amino acid sequencecomprising the following domains in the indicated order: (i) atranscription factor (TF) domain comprising a sequence from animmunosuppressive human immunodeficiency virus (HIV) or simianimmunodeficiency virus (SIV) transactivator of transcription (Tat)protein, hairless or an artificial immunosuppressive sequence, whereinthe TF domain comprises the amino acid sequence of one of SEQ ID NOs:36, 39, 44, 48, 50, or 54; (ii) a cysteine-rich region from lentiviralTat or a defensin molecule, wherein the cysteine-rich region comprisesthe amino acid sequence of one of SEQ ID NOs: 37, 40, 41, 43, 45, 51,55, 57, 58, 62, 63, 64, or 70; and (iii) a C-terminal region from alentiviral Tat protein comprising the amino acid sequence of one of SEQID NOs: 38, 42, 46, 52, 68, or 71; (B) a transcription factor (TF)domain according to (i), and a cysteine-rich domain and C-terminaldomain, in that order, wherein the cysteine-rich domain and C-terminaldomain together comprise an amino acid sequence of one of SEQ ID NOs:47, 49 or 53; or (C) or a conservative variant of the immunosuppressivepolypeptide of (A) or (B) having at least 95% sequence identity thereto;and thereby increasing reducing inflammation in the subject.